Genes and uses for plant improvement

ABSTRACT

Transgenic seed for crops with improved traits are provided by trait-improving recombinant DNA in the nucleus of cells of the seed where plants grown from such transgenic seed exhibit one or more improved traits as compared to a control plant. Of particular interest are transgenic plants that have increased yield. The present invention also provides recombinant DNA molecules for expression of a protein, and recombinant DNA molecules for suppression of a protein.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims benefit under 35USC §119(e) of U.S. provisional application Ser. No. 60/873,247, filed Dec. 6, 2006, herein incorporated by reference.

INCORPORATION OF SEQUENCE LISTING

Two copies of the sequence listing (Copy 1 and Copy 2) and a computer readable form (CRF) of the sequence listing, all on CD-Rs, each containing the file named 38-21(53949)B_seqListing.txt, which is 67,317,760 bytes (measured in MS-WINDOWS) and was created on Dec. 4, 2007, are incorporated herein by reference in their entirety.

INCORPORATION OF COMPUTER PROGRAM LISTING

A Computer Program Listing with folders “hmmer-2.3.2” and “161pfamDir” is contained on a CD-R and is incorporated herein by reference in their entirety. Folder hmmer-2.3.2 contains the source code and other associated file for implementing the HMMer software for Pfam analysis. Folder 161pfamDir contains 161 profile Hidden Markov Models. Both folders were created on the disk on Dec. 4, 2007 having a total size of 15,534,080 bytes when measured in MS-WINDOWS® operating system.

FIELD OF THE INVENTION

Disclosed herein are transgenic plant cells, plants and seeds comprising recombinant DNA and methods of making and using such plant cells, plants and seeds

BACKGROUND OF THE INVENTION

Transgenic plants with enhanced traits such as improved yield, environmental stress tolerance, pest resistance, herbicide tolerance, modified seed compositions, and the like are desired by both farmers and consumers. Although considerable efforts in plant breeding have provided significant gains in desired traits, the ability to introduce specific DNA into plant genomes provides further opportunities for generation of plants with improved and/or unique traits. The ability to develop transgenic plants with improved traits depends in part on the identification of useful recombinant DNA for production of transformed plants with improved properties, e.g. by actually selecting a transgenic plant from a screen for such improved property. An object of this invention is to provide transgenic plant cell nuclei, plant cells, plants and seeds by screening transgenic crop plants for one of more enhanced agronomic traits where the nucleus in cells of the plant or seed has recombinant DNA that was identified as imparting an improved trait in a model plant, e.g. Arabidopsis thaliana. In some cases the model plant may exhibit an improved trait that corresponds to an enhanced agronomic trait, e.g. cold stress tolerance, water deficit stress tolerance, low nitrogen stress tolerance and the like. In other cases the model plant may exhibit an improved trait that is a surrogate to an enhanced agronomic trait, e.g. salinity stress tolerance being a surrogate to drought tolerance or improvement in plant growth and development being a surrogate to enhanced yield. A further object of the invention is to provide screening methods requiring routine experimentation by which such transgenic plant cell nuclei, cells, plants and seeds can be identified by making a reasonable number of transgenic events and engaging in screening identified in this specification and illustrated in the examples.

SUMMARY OF THE INVENTION

This invention provides plant cell nuclei with recombinant DNA that imparts enhanced agronomic traits in transgenic plants having the nuclei in their cells. Recombinant DNA in this invention is provided in a construct comprising a promoter that is functional in plant cells and that is operably linked to DNA that encodes a protein having at least one amino acid domain in a sequence that exceeds the Pfam gathering cutoff for amino acid sequence alignment with a protein domain family identified by a Pfam name in the group of Pfam domain names identified in Table 17. In more specific embodiments of the invention plant cells are provided which express a protein having amino acid sequence with at least 90% identity to a consensus amino acid sequence in the group of consensus amino acid sequences consisting of the consensus amino acid sequence constructed for SEQ ID NO: 198 and homologs thereof listed in Table 2 through the consensus amino acid sequence constructed for SEQ ID NO: 394 and homologs thereof listed in Table 2. Amino acid sequences of homologs are SEQ ID NO: 395 through 19,938. In even more specific embodiments of the invention the protein expressed in plant cells is a protein selected from the group of proteins identified in Table 1 by annotation to a related protein in Genbank and alternatively identified in Table 16 by identification of protein domain family.

Other aspects of the invention are specifically directed to transgenic plant cells, and transgenic plants comprising a plurality of plant cells with such nuclei, progeny transgenic seed, embryo and transgenic pollen from such plants. Such plant cell nuclei are selected from a population of transgenic plants regenerated from plant cells with a nucleus transformed with recombinant DNA by screening the transgenic plants in the population for an enhanced trait as compared to control plants that do not have the recombinant DNA in their nucleus, where the enhanced trait is enhanced water use efficiency, enhanced cold tolerance, enhanced heat tolerance, enhanced shade tolerance, enhanced tolerance to salt exposure, increased yield, enhanced nitrogen use efficiency, enhanced seed protein or enhanced seed oil. In some aspects of the invention the recombinant DNA expresses a protein that imparts the enhanced trait; in other aspects of the invention the recombinant DNA expresses RNA for suppressing the level of an endogenous protein. In yet another aspect of the invention the nucleus of plant cells in plants, seeds, embryo and pollen further comprise DNA expressing a protein that provides tolerance from exposure to an herbicide applied at levels that are lethal to a wild type plant cell. Such tolerance is especially useful not only as an advantageous trait in such plants but is also useful in a selection step in the methods of the invention. In aspects of the invention the agent of such herbicide is a glyphosate, dicamba, or glufosinate compound.

Yet other aspects of the invention provide nuclei in cells of transgenic plants which are homozygous for the recombinant DNA and transgenic seed of the invention from corn, soybean, cotton, canola, alfalfa, wheat or rice plants.

In other embodiments for practice of various aspects of the invention in Argentina the recombinant DNA in the nucleus is provided in plant cells derived from corn lines that are and maintain resistance to a virus such as the Mal de Rio Cuarto virus or a fungus such as the Puccina sorghi fungus or to both.

This invention also provides methods for manufacturing non-natural, transgenic seed that can be used to produce a crop of transgenic plants with an enhanced trait resulting from expression of stably-integrated, recombinant DNA in the nucleus of the plant cells. In some aspects of the invention the recombinant DNA can express a protein having at least one domain of amino acids in a sequence that exceeds the Pfam gathering cutoff for amino acid sequence alignment with a protein domain family identified by a Pfam name in the group of Pfam names identified in Table 17; in other aspects the recombinant DNA suppresses the level of such a protein. More specifically the method comprises (a) screening a population of plants for an enhanced trait and recombinant DNA, where individual plants in the population can exhibit the trait at a level less than, essentially the same as or greater than the level that the trait is exhibited in control plants which do not express the recombinant DNA; (b) selecting from the population one or more plants that exhibit the trait at a level greater than the level that said trait is exhibited in control plants; (c) verifying that the recombinant DNA is stably integrated in said selected plants; (d) analyzing tissue of a selected plant to determine the production of a protein having the function of a protein encoded by nucleotides in a sequence of one of SEQ ID NO: 1-197; and (e) collecting seed from a selected plant. In one aspect of the invention the plants in the population further comprise DNA expressing a protein that provides tolerance to exposure to an herbicide applied at levels that are lethal to wild type plant cells and where the selecting is effected by treating the population with the herbicide, e.g. a glyphosate, dicamba, or glufosinate compound. In another aspect of the invention the plants are selected by identifying plants with the enhanced trait. The methods are especially useful for manufacturing corn, soybean, cotton, alfalfa, wheat or rice seed selected as having at least one of the enhanced traits described above.

Another aspect of the invention provides a method of producing hybrid corn seed comprising acquiring hybrid corn seed from a herbicide tolerant corn plant which also has a nucleus of this invention with stably-integrated, recombinant DNA The method further comprises producing corn plants from said hybrid corn seed, where a fraction of the plants produced from said hybrid corn seed is homozygous for said recombinant DNA, a fraction of the plants produced from said hybrid corn seed is hemizygous for said recombinant DNA, and a fraction of the plants produced from said hybrid corn seed has none of said recombinant DNA; selecting corn plants which are homozygous and hemizygous for said recombinant DNA by treating with an herbicide; collecting seed from herbicide-treated-surviving corn plants and planting said seed to produce further progeny corn plants; repeating the selecting and collecting steps at least once to produce an inbred corn line; and crossing the inbred corn line with a second corn line to produce hybrid seed.

Another aspect of the invention provides a method of selecting a plant comprising a nucleus of this invention in its plant cells by using an immunoreactive antibody to detect the presence of protein expressed by recombinant DNA in seed or plant tissue. Another aspect of the invention provides anti-counterfeit milled seed having, as an indication of origin, a nucleus of this invention with unique recombinant DNA.

Aspects of the invention relating to nucleus in plant cells having recombinant DNA for suppressing the expression of a protein are identified in Table 1 and Table 16. More specific aspects of the invention provide plant cells having recombinant DNA for suppressing the expression of a protein having the function in a plant of the protein with amino acid sequence of SEQ ID NO: 200, 201, 205, 207, 211 and 394 or the corresponding Pfam identified in Table 16, i.e. SNF5, LMBR1, TFIIS_M, TFIIS_C, Glyco_transf_(—)8, respectively. Such suppression can be effected by any of a number of ways known in the art, e.g. anti-sense suppression, RNAi or mutation knockout and the like.

Another aspect of this invention relates to growing transgenic plants with enhanced water use efficiency or enhanced nitrogen use efficiency. For instance, this invention provides methods of growing a corn, cotton or soybean crop without irrigation water comprising planting seed having plant cells of the invention which are selected for enhanced water use efficiency. Alternatively methods comprise applying reduced irrigation water, e.g. providing up to 300 millimeters of ground water during the production of a corn crop. This invention also provides methods of growing a corn, cotton or soybean crop without added nitrogen fertilizer comprising planting seed having plant cells of the invention which are selected for enhanced nitrogen use efficiency. Alternatively methods comprise applying reduced amount of nitrogen input as compared to the conventional input during the production of a corn crop.

The various aspects of this invention are especially useful for transgenic plant cells in seeds and transgenic plants having any of the above-described enhanced traits in crop plants such as corn (maize), soybean, cotton, canola (rape), wheat, sunflower, sorghum, alfalfa, barley, millet, rice, tobacco, fruit and vegetable crops, and turfgrass.

The invention also provides recombinant DNA constructs comprising the DNA useful in the nuclei in plant cells for imparting enhanced traits in plants having those cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a consensus amino acid sequence of SEQ ID NO: 227 and its homologs.

FIGS. 2, 3 and 4 illustrate plasmid maps.

DETAILED DESCRIPTION OF THE INVENTION

In the attached sequence listing:

SEQ ID NO: 1-197 are nucleotide sequences of the coding strand of DNA for “genes” used in the recombinant DNA imparting an enhanced trait in plant cells, i.e. each represents a coding sequence for a protein;

SEQ ID NO: 198-394 are amino acid sequences of the cognate protein of the “genes” with nucleotide coding sequence 1-197;

SEQ ID NO: 395-19938 are amino acid sequences of homologous proteins;

SEQ ID NO:_(—)19939 is a consensus amino acid sequence.

SEQ ID NO: 19940 is a nucleotide sequence of a plasmid base vector useful for corn transformation; and

SEQ ID NO: 19941 is a DNA sequence of a plasmid base vector useful for soybean or canola transformation.

SEQ ID NO:19942 is a DNA sequence of a plasmid base vector useful for cotton transformation.

The nuclei of this invention are identified by screening transgenic plants for one or more traits including improved drought stress tolerance, improved heat stress tolerance, improved cold stress tolerance, improved high salinity stress tolerance, improved low nitrogen availability stress tolerance, improved shade stress tolerance, improved plant growth and development at the stages of seed imbibition through early vegetative phase, and improved plant growth and development at the stages of leaf development, flower production and seed maturity.

“Gene” refers to chromosomal DNA, plasmid DNA, cDNA, synthetic DNA, or other DNA that encodes a peptide, polypeptide, protein, or RNA molecule, and regions flanking the coding sequences involved in the regulation of expression. In aspects of the invention where an improved trait is provided by expression of a protein, “gene” refers at least to coding nucleotide sequence for a protein or a functional polypeptide fragment of a protein that imparts the trait. In aspects of the invention where an improved trait is provided by suppression of expression of an endogenous protein, “gene” refers to any part of the gene that can be a target for suppression.

“Transgenic seed” means a plant seed whose nucleus has been altered by the incorporation of recombinant DNA, e.g., by transformation as described herein. The term “transgenic plant” is used to refer to the plant produced from an original transformation event, or progeny from later generations or crosses of a plant to a transformed plant, so long as the progeny contains a nucleus with the recombinant DNA in its genome.

“Recombinant DNA” means a polynucleotide having a genetically engineered modification introduced through combination of endogenous and/or exogenous elements in a transcription unit, manipulation via mutagenesis, restriction enzymes, and the like or simply by inserting multiple copies of a native transcription unit. Recombinant DNA may comprise DNA segments obtained from different sources, or DNA segments obtained from the same source, but which have been manipulated to join DNA segments which do not naturally exist in the joined form. A recombinant polynucleotide may exist outside of the cell, for example as a PCR fragment, or integrated into a genome, such as a plant genome.

“Trait” means a physiological, morphological, biochemical, or physical characteristic of a plant or particular plant material or cell. In some instances, this characteristic is visible to the human eye, such as seed or plant size, or can be measured by biochemical techniques, such as detecting the protein, starch, or oil content of seed or leaves, or by observation of a metabolic or physiological process, e.g., by measuring uptake of carbon dioxide, or by the observation of the expression level of a gene or genes, e.g., by employing Northern analysis, RT-PCR, microarray gene expression assays, or reporter gene expression systems, or by agricultural observations such as stress tolerance, yield, or pathogen tolerance.

A “control plant” is a plant without trait-improving recombinant DNA in its nucleus. A control plant is used to measure and compare trait improvement in a transgenic plant with such trait-improving recombinant DNA. A suitable control plant may be a non-transgenic plant of the parental line used to generate a transgenic plant herein. Alternatively, a control plant may be a transgenic plant that comprises an empty vector or marker gene, but does not contain the recombinant DNA that produces the trait improvement. A control plant may also be a negative segregant progeny of hemizygous transgenic plant. In certain demonstrations of trait improvement, the use of a limited number of control plants can cause a wide variation in the control dataset. To minimize the effect of the variation within the control dataset, a “reference” is used. As use herein a “reference” is a trimmed mean of all data from both transgenic and control plants grown under the same conditions and at the same developmental stage. The trimmed mean is calculated by eliminating a specific percentage, e.g., 20%, of the smallest and largest observation from the data set and then calculating the average of the remaining observation.

“Trait enhancement” means a detectable and desirable difference in a characteristic in a transgenic plant relative to a control plant or a reference. In some cases, the trait enhancement can be measured quantitatively. For example, the trait improvement can entail at least a 2% desirable difference in an observed trait, at least a 5% desirable difference, at least about a 10% desirable difference, at least about a 20% desirable difference, at least about a 30% desirable difference, at least about a 50% desirable difference, at least about a 70% desirable difference, or at least about a 100% difference, or an even greater desirable difference. In other cases, the trait enhancement is only measured qualitatively. It is known that there can be a natural variation in a trait. Therefore, the trait enhancement observed entails a change of the normal distribution of the trait in the transgenic plant compared with the trait distribution observed in a control plant or a reference, which is evaluated by statistical methods provided herein. Trait enhancement includes, but is not limited to, yield increase, including increased yield under non-stress conditions and increased yield under environmental stress conditions. Stress conditions may include, for example, drought, shade, fungal disease, viral disease, bacterial disease, insect infestation, nematode infestation, cold temperature exposure, heat exposure, osmotic stress, reduced nitrogen nutrient availability, reduced phosphorus nutrient availability and high plant density.

Many agronomic traits can affect “yield”, including without limitation, plant height, pod number, pod position on the plant, number of internodes, incidence of pod shatter, grain size, efficiency of nodulation and nitrogen fixation, efficiency of nutrient assimilation, resistance to biotic and abiotic stress, carbon assimilation, plant architecture, resistance to lodging, percent seed germination, seedling vigor, and juvenile traits. Other traits that can affect yield include, efficiency of germination (including germination in stressed conditions), growth rate (including growth rate in stressed conditions), ear number, seed number per ear, seed size, composition of seed (starch, oil, protein) and characteristics of seed fill. Also of interest is the generation of transgenic plants that demonstrate desirable phenotypic properties that may or may not confer an increase in overall plant yield. Such properties include enhanced plant morphology, plant physiology or improved components of the mature seed harvested from the transgenic plant.

“Yield-limiting environment” means the condition under which a plant would have the limitation on yield including environmental stress conditions.

“Stress condition” means a condition unfavorable for a plant, which adversely affect plant metabolism, growth and/or development. A plant under the stress condition typically shows reduced germination rate, retarded growth and development, reduced photosynthesis rate, and eventually leading to reduction in yield. Specifically, “water deficit stress” used herein preferably refers to the sub-optimal conditions for water and humidity needed for normal growth of natural plants. Relative water content (RWC) can be used as a physiological measure of plant water deficit. It measures the effect of osmotic adjustment in plant water status, when a plant is under stress conditions. Conditions that may result in water deficit stress include heat, drought, high salinity and PEG induced osmotic stress.

“Cold stress” means the exposure of a plant to a temperatures below (two or more degrees Celsius below) those normal for a particular species or particular strain of plant.

“Nitrogen nutrient” means any one or any mix of the nitrate salts commonly used as plant nitrogen fertilizer, including, but not limited to, potassium nitrate, calcium nitrate, sodium nitrate, ammonium nitrate. The term ammonium as used herein means any one or any mix of the ammonium salts commonly used as plant nitrogen fertilizer, e.g., ammonium nitrate, ammonium chloride, ammonium sulfate, etc.

“Low nitrogen availability stress” means a plant growth condition that does not contain sufficient nitrogen nutrient to maintain a healthy plant growth and/or for a plant to reach its typical yield under a sufficient nitrogen growth condition. For example, a low nitrogen condition can refers to a growth condition with 50% or less of the conventional nitrogen inputs. “Sufficient nitrogen growth condition” means a growth condition where the soil or growth medium contains or receives optimal amounts of nitrogen nutrient to sustain a healthy plant growth and/or for a plant to reach its typical yield for a particular plant species or a particular strain. One skilled in the art would recognize what constitute such soil, media and fertilizer inputs for most plant species.

“Shade stress” means a growth condition that has limited light availability that triggers the shade avoidance response in plant. Plants are subject to shade stress when localized at lower part of the canopy, or in close proximity of neighboring vegetation. Shade stress may become exacerbated when the planting density exceeds the average prevailing density for a particular plant species. The average prevailing densities per acre of a few examples of crop plants in the USA in the year 2000 were: wheat 1,000,000-1,500,000; rice 650,000-900,000; soybean 150,000-200,000, canola 260,000-350,000, sunflower 17,000-23,000 and cotton 28,000-55,000 plants per acre (Cheikh, e.g., (2003) U.S. Patent Application No. 20030101479).

“Increased yield” of a transgenic plant of the present invention is evidenced and measured in a number of ways, including test weight, seed number per plant, seed weight, seed number per unit area (i.e., seeds, or weight of seeds, per acre), bushels per acre, tons per acre, tons per acre, kilo per hectare. For example, maize yield can be measured as production of shelled corn kernels per unit of production area, e.g., in bushels per acre or metric tons per hectare, often reported on a moisture adjusted basis, e.g., at 15.5% moisture. Increased yield can result from improved utilization of key biochemical compounds, such as nitrogen, phosphorous and carbohydrate, or from improved tolerance to environmental stresses, such as cold, heat, drought, salt, and attack by pests or pathogens. Trait-improving recombinant DNA can also be used to provide transgenic plants having improved growth and development, and ultimately increased yield, as the result of modified expression of plant growth regulators or modification of cell cycle or photosynthesis pathways.

“Expression” means transcription of DNA to produce RNA. The resulting RNA may be without limitation mRNA encoding a protein, antisense RNA, or a double-stranded RNA for use in RNAi technology. Expression also refers to production of encoded protein from mRNA.

A “plant promoter” is a promoter capable of initiating transcription in plant cells whether or not its origin is a plant cell. Exemplary plant promoters include, but are not limited to, those that are obtained from plants, plant viruses, and bacteria which comprise genes expressed in plant cells such Agrobacterium or Rhizobium. Examples of promoters under developmental control include promoters that preferentially initiate transcription in certain tissues, such as leaves, roots, or seeds. Such promoters are referred to as “tissue preferred”. Promoters which initiate transcription only in certain tissues are referred to as “tissue specific”. A “cell type” specific promoter primarily drives expression in certain cell types in one or more organs, for example, vascular cells in roots or leaves. An “inducible” or “repressible” promoter is a promoter which is under environmental control. Examples of environmental conditions that may effect transcription by inducible promoters include anaerobic conditions, or certain chemicals, or the presence of light. Tissue specific, tissue preferred, cell type specific, and inducible promoters constitute the class of “non-constitutive” promoters. A “constitutive” promoter is a promoter which is active under most conditions. As used herein, “antisense orientation” includes reference to a polynucleotide sequence that is operably linked to a promoter in an orientation where the antisense strand is transcribed. The antisense strand is sufficiently complementary to an endogenous transcription product such that translation of the endogenous transcription product is often inhibited.

As used herein, “operably linked” refers to the association of two or more nucleic acid fragments on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.

A “consensus sequence” refers to an artificial, amino acid sequence of conserved parts of the proteins encoded by homologous genes, e.g., as determined by a CLUSTALW alignment of amino acid sequence of homolog proteins.

Homologous genes are genes which encode proteins with the same or similar biological function to the protein encoded by the second gene. Homologous genes may be generated by the event of speciation (see ortholog) or by the event of genetic duplication (see paralog). “Orthologs” refer to a set of homologous genes in different species that evolved from a common ancestral gene by specification. Normally, orthologs retain the same function in the course of evolution; and “paralogs” refer to a set of homologous genes in the same species that have diverged from each other as a consequence of genetic duplication. Thus, homologous genes can be from the same or a different organism. As used herein, “homolog” means a protein that performs the same biological function as a second protein including those identified by sequence identity search.

Percent identity refers to the extent to which two optimally aligned DNA or protein segments are invariant throughout a window of alignment of components, e.g., nucleotide sequence or amino acid sequence. An “identity fraction” for aligned segments of a test sequence and a reference sequence is the number of identical components which are shared by sequences of the two aligned segments divided by the total number of sequence components in the reference segment over a window of alignment which is the smaller of the full test sequence or the full reference sequence. “Percent identity” (“% identity”) is the identity fraction times 100. “% identity to a consensus amino acid sequence” is 100 times the identity fraction in a window of alignment of an amino acid sequence of a test protein optimally aligned to consensus amino acid sequence of this invention.

“Arabidopsis” means plants of Arabidopsis thaliana.

“Pfam” refers to a large collection of multiple sequence alignments and hidden Markov models covering many common protein families, e.g. Pfam version 18.0 (August 2005) contains alignments and models for 7973 protein families and is based on the Swissprot 47.0 and SP-TREMBL 30.0 protein sequence databases. See S. R. Eddy, “Profile Hidden Markov Models”, Bioinformatics 14:755-763, 1998. Pfam is currently maintained and updated by a Pfam Consortium. The alignments represent some evolutionary conserved structure that has implications for the protein's function. Profile hidden Markov models (profile HMMs) built from the Pfam alignments are useful for automatically recognizing that a new protein belongs to an existing protein family even if the homology by alignment appears to be low. Once one DNA is identified as encoding a protein which imparts an enhanced trait when expressed in transgenic plants, other DNA encoding proteins in the same protein family are identified by querying the amino acid sequence of protein encoded by candidate DNA against the Hidden Markov Model which characterizes the Pfam domain using HMMER software, a current version of which is provided in the appended computer listing. Candidate proteins meeting the gathering cutoff for the alignment of a particular Pfam are in the protein family and have cognate DNA that is useful in constructing recombinant DNA for the use in the plant cells of this invention. Hidden Markov Model databases for use with HMMER software in identifying DNA expressing protein in a common Pfam for recombinant DNA in the plant cells of this invention are also included in the appended computer listing. The HMMER software and Pfam databases are version 18.0 and were used to identify known domains in the proteins corresponding to amino acid sequence of SEQ ID NO: 198 through SEQ ID NO: 394. All DNA encoding proteins that have scores higher than the gathering cutoff disclosed in Table 17 by Pfam analysis disclosed herein can be used in recombinant DNA of the plant cells of this invention, e.g. for selecting transgenic plants having enhanced agronomic traits. The relevant Pfams for use in this invention, as more specifically disclosed below, are L51_S25_CI-B8, iPGM_N, WD40, BPL_LipA_LipB, DUF676, AAA, S_locus_glycop, ArfGap, Rotamase, Metallophos, CMAS, Sugar_tr, LMBR1, RrnaAD, NAF, BolA, Pkinase, C2, FA_hydroxylase, p450, Complex1_(—)30 kDa, Histone, DUF822, PEP-utilizers, PCI, ETC_C1_NDUFA5, 2-Hacid_dh, Tryp_alpha_amyl, PK_C, MAP65_ASE1, FBPase, SWIB, Ank, Ribosomal_S8e, 2-Hacid_dh_C, SMC_N, GTP_cyclohydro-2, PfkB, ORMDL, ADH_zinc_N, SWIM, TrkA_N, HLH, GH3, SNF5, Ceramidase_alk, Ribonuclease_T2, Complex1_(—)49 kDa, Gp_dh_C, Aldo_ket_red, zf-AN1, TFIIS_C, MFS_(—)1, Thioredoxin, DUF1005, LEA_(—)3, Sterol_MT_C, Gp_dh_N, TFIIS_M, PAN_(—)2, BPL_C, DUF26, Aa_trans, ACT, ADH_N, NAD_binding_(—)1, Auxin_inducible, B_lectin, Anti-silence, Response_reg, 14-3-3, LRRNT_(—)2, GDC-P, zf-CCHC, NPH3, TPR_(—)1, TFIIA, DHBP_synthase, UQ_con, TPR_(—)2, TPT, F-box, adh_short, Cyclin_C, Na_H_Exchanger, AA_permease, MtN3_slv, TIM, NDK, Pantoate_transf, Allene_ox_cyc, Cyclin_N, Methyltransf_(—)11, CBM_(—)20, Methyltransf_(—)12, Rhodanese, Glycolytic, Actin, Usp, eIF-4B, Glyco_transf 8, BURP, Alpha-amylase, F420_oxidored, EGF_CA, Kelch_(—)1, PGAM, Aminotran_(—)1_(—)2, Kelch_(—)2, UPF0261, CoA_binding, DUF868, Peptidase_S10, Lung_(—)7-TM_R, Oleosin, Sad1_UNC, Gln-synt_C, LSM, NTP_transferase, Metalloenzyme, Prenylcys_lyase, Subtilisin_N, SAM_(—)1, DUF298, ESCRT-III, DNA_pol_E_B, Aminotran_(—)3, NAD_Gly3P_dh_N, Gln-synt_N, MMR_HSR1, DUF588, zf-CCCH, DnaJ, Pkinase_Tyr, Cupin_(—)2, LRR_(—)1, Cupin_(—)3, zf-CSL, FAR1, HD, FH2, APC8, PTR₂, MannoseP_isomer, Rib_(—)5-P_isom_A, DUF1336, Phosphorylase, DUF1191, Asp, Mit_rib_S27, PAP_fibrillin, DUF1195, Aldedh, zf-C3HC4, PPR, PK, PurA, RMMBL, HTH_(—)11, Tim17, and PBD.

Recombinant DNA Constructs

The present invention provides recombinant DNA constructs comprising one or more polynucleotides disclosed herein for imparting one or more improved traits to transgenic plant when incorporated into the nucleus of the plant cells. Such constructs also typically comprise a promoter operatively linked to said polynucleotide to provide for expression in the plant cells. Other construct components may include additional regulatory elements, such as 5′ or 3′ untranslated regions (such as polyadenylation sites), intron regions, and transit or signal peptides. Such recombinant DNA constructs can be assembled using methods known to those of ordinary skill in the art.

In a preferred embodiment, a polynucleotide of the present invention is operatively linked in a recombinant DNA construct to a promoter functional in a plant to provide for expression of the polynucleotide in the sense orientation such that a desired protein or polypeptide fragment of a protein is produced. Also provided are embodiments wherein a polynucleotide is operatively linked to a promoter functional in a plant to provide for expression of gene suppression RNA to suppress the level of an endogenous protein.

Recombinant constructs prepared in accordance with the present invention also generally include a 3′ untranslated DNA region (UTR) that typically contains a polyadenylation sequence following the polynucleotide coding region. Examples of useful 3′ UTRs include those from the nopaline synthase gene of Agrobacterium tumefaciens (nos), a gene encoding the small subunit of a ribulose-1,5-bisphosphate carboxylase-oxygenase (rbcS), and the T7 transcript of Agrobacterium tumefaciens.

Constructs and vectors may also include a transit peptide for targeting of a gene target to a plant organelle, particularly to a chloroplast, leucoplast or other plastid organelle. For descriptions of the use of chloroplast transit peptides, see U.S. Pat. No. 5,188,642 and U.S. Pat. No. 5,728,925, incorporated herein by reference.

Tablet provides a list of genes that provided recombinant DNA that was expressed in a model plant and identified from screening as imparting an improved trait. When the stated orientation is “sense”, the expression of the gene or a homolog in a crop plant provides the means to identify transgenic events that provide an enhanced trait in the crop plant. When the stated orientation is “antisense”, the suppression of the native homolog in a crop plant provides the means to identify transgenic events that provide an enhanced trait in the crop plant. In some cases the expression/suppression in the model plant exhibited an improved trait that corresponds to an enhanced agronomic trait, e.g. cold stress tolerance, water deficit stress tolerance, low nitrogen stress tolerance and the like. In other cases the expression/suppression in the model plant exhibited an improved trait that is a surrogate to an enhance agronomic trait, e.g. salinity stress tolerance being a surrogate to drought tolerance or improvement in plant growth and development being a surrogate to enhanced yield. Even when expression of a transgene or suppression of a native gene imparts an improved trait in a model plant, not every crop plant expressing the same transgene or suppressing the same native gene will necessarily demonstrate an indicated enhanced agronomic trait. For instance, it is well known that multiple transgenic events are required to identify a transgenic plant that can exhibit an enhanced agronomic trait. However, by with routine experimentation a transgenic plant cell nuclei, cell, plant or seed of this invention can be identified by making a reasonable number of transgenic events and engaging in screening process identified in this specification and illustrated in the examples. An understanding of Table 1 is facilitated by the following description of the headings:

“NUC SEQ ID NO” refers to a SEQ ID NO. for particular DNA sequence in the Sequence Listing.

“PEP SEQ ID NO” refers to a SEQ ID NO. in the Sequence Listing for the amino acid sequence of a protein cognate to a particular DNA

“construct_id” refers to an arbitrary number used to identify a particular recombinant DNA construct comprising the particular DNA.

“Gene ID” refers to an arbitrary name used to identify the particular DNA.

“orientation” refers to the orientation of the particular DNA in a recombinant DNA construct relative to the promoter.

TABLE 1 NUC PEP Seq SEQ ID ID Construct No. No Gene ID ID Orientation 1 198 CGPG106 11029 SENSE 2 199 CGPG1133 12223 SENSE 3 200 CGPG117 10422 ANTI-SENSE 4 201 CGPG1226 13485 ANTI-SENSE 5 202 CGPG1288 13235 SENSE 6 203 CGPG1301 13411 SENSE 7 204 CGPG1458 73944 SENSE 8 205 CGPG1542 13846 ANTI-SENSE 9 206 CGPG170 12602 SENSE 10 207 CGPG1828 74065 ANTI-SENSE 11 208 CGPG2206 72783 SENSE 12 209 CGPG2217 17210 SENSE 13 210 CGPG2292 72724 SENSE 14 211 CGPG2457 17805 ANTI-SENSE 15 212 CGPG2499 16610 SENSE 16 213 CGPG2653 76602 SENSE 17 214 CGPG2813 18456 SENSE 18 215 CGPG3002 18414 SENSE 19 216 CGPG3154 71538 SENSE 20 217 CGPG3235 76532 SENSE 21 218 CGPG3274 18231 SENSE 22 219 CGPG3275 18232 SENSE 23 220 CGPG3363 18256 SENSE 24 221 CGPG3367 18258 SENSE 25 222 CGPG3375 19193 SENSE 26 223 CGPG3528 71301 SENSE 27 224 CGPG3534 18354 SENSE 28 225 CGPG3638 77334 SENSE 29 226 CGPG3918 19767 SENSE 30 227 CGPG3920 19774 SENSE 31 228 CGPG3962 70992 SENSE 32 229 CGPG3972 19956 SENSE 33 230 CGPG3990 70948 SENSE 34 231 CGPG3994 70201 SENSE 35 232 CGPG4026 19973 SENSE 36 233 CGPG4048 70987 SENSE 37 234 CGPG4052 70950 SENSE 38 235 CGPG4057 70962 SENSE 39 236 CGPG4058 70915 SENSE 40 237 CGPG4069 19947 SENSE 41 238 CGPG4087 70969 SENSE 42 239 CGPG4088 70985 SENSE 43 240 CGPG4102 70971 SENSE 44 241 CGPG4121 70963 SENSE 45 242 CGPG4122 70994 SENSE 46 243 CGPG4140 70956 SENSE 47 244 CGPG4154 70995 SENSE 48 245 CGPG4311 73306 SENSE 49 246 CGPG4363 70657 SENSE 50 247 CGPG4369 70660 SENSE 51 248 CGPG442 74536 SENSE 52 249 CGPG4454 71328 SENSE 53 250 CGPG4456 71329 SENSE 54 251 CGPG4473 70755 SENSE 55 252 CGPG4588 70684 SENSE 56 253 CGPG4765 73330 SENSE 57 254 CGPG4788 76202 SENSE 58 255 CGPG4912 72807 SENSE 59 256 CGPG4926 72811 SENSE 60 257 CGPG4967 73235 SENSE 61 258 CGPG4977 72813 SENSE 62 259 CGPG5001 72825 SENSE 63 260 CGPG5025 73628 SENSE 64 261 CGPG5041 76105 SENSE 65 262 CGPG5116 73242 SENSE 66 263 CGPG5144 74217 SENSE 67 264 CGPG5171 73735 SENSE 68 265 CGPG5194 73256 SENSE 69 266 CGPG5200 73260 SENSE 70 267 CGPG5210 75822 SENSE 71 268 CGPG5221 72001 SENSE 72 269 CGPG5269 72056 SENSE 73 270 CGPG5404 77308 SENSE 74 271 CGPG5432 73766 SENSE 75 272 CGPG5518 72774 SENSE 76 273 CGPG5535 72788 SENSE 77 274 CGPG5540 72753 SENSE 78 275 CGPG5568 72709 SENSE 79 276 CGPG5577 73954 SENSE 80 277 CGPG5587 73137 SENSE 81 278 CGPG5594 73161 SENSE 82 279 CGPG5633 73057 SENSE 83 280 CGPG5640 73127 SENSE 84 281 CGPG5646 73033 SENSE 85 282 CGPG5656 73105 SENSE 86 283 CGPG5659 73141 SENSE 87 284 CGPG5661 73165 SENSE 88 285 CGPG5684 73155 SENSE 89 286 CGPG5694 73026 SENSE 90 287 CGPG5704 73120 SENSE 91 288 CGPG5714 73133 SENSE 92 289 CGPG5721 73134 SENSE 93 290 CGPG5728 73123 SENSE 94 291 CGPG5757 73981 SENSE 95 292 CGPG5764 73136 SENSE 96 293 CGPG5783 73172 SENSE 97 294 CGPG5791 73020 SENSE 98 295 CGPG5799 72946 SENSE 99 296 CGPG5856 74746 SENSE 100 297 CGPG5927 77312 SENSE 101 298 CGPG5941 75237 SENSE 102 299 CGPG5957 75240 SENSE 103 300 CGPG5967 74349 SENSE 104 301 CGPG6040 76422 SENSE 105 302 CGPG607 70812 SENSE 106 303 CGPG6178 77322 SENSE 107 304 CGPG6185 74662 SENSE 108 305 CGPG6306 76527 SENSE 109 306 CGPG6318 77020 SENSE 110 307 CGPG6326 77609 SENSE 111 308 CGPG6370 73485 SENSE 112 309 CGPG6429 73433 SENSE 113 310 CGPG6440 73411 SENSE 114 311 CGPG6516 73568 SENSE 115 312 CGPG6653 74688 SENSE 116 313 CGPG6712 74420 SENSE 117 314 CGPG6737 74435 SENSE 118 315 CGPG6747 74460 SENSE 119 316 CGPG6796 74566 SENSE 120 317 CGPG6805 77610 SENSE 121 318 CGPG6810 77618 SENSE 122 319 CGPG6952 77517 SENSE 123 320 CGPG6953 77518 SENSE 124 321 CGPG7121 76460 SENSE 125 322 CGPG7163 77069 SENSE 126 323 CGPG7168 76161 SENSE 127 324 CGPG7206 76171 SENSE 128 325 CGPG7225 76178 SENSE 129 326 CGPG7267 76467 SENSE 130 327 CGPG7272 77536 SENSE 131 328 CGPG7281 76576 SENSE 132 329 CGPG7308 74862 SENSE 133 330 CGPG7316 74863 SENSE 134 331 CGPG7371 74858 SENSE 135 332 CGPG7457 74933 SENSE 136 333 CGPG7520 75379 SENSE 137 334 CGPG7529 77816 SENSE 138 335 CGPG7636 75434 SENSE 139 336 CGPG7737 77821 SENSE 140 337 CGPG7767 75685 SENSE 141 338 CGPG7804 75654 SENSE 142 339 CGPG7823 75692 SENSE 143 340 CGPG7828 75657 SENSE 144 341 CGPG7833 75622 SENSE 145 342 CGPG7933 77549 SENSE 146 343 CGPG7986 77917 SENSE 147 344 CGPG8012 77568 SENSE 148 345 CGPG8015 77570 SENSE 149 346 CGPG8055 77338 SENSE 150 347 CGPG8062 77580 SENSE 151 348 CGPG8082 77928 SENSE 152 349 CGPG8083 77349 SENSE 153 350 CGPG8106 77357 SENSE 154 351 CGPG8107 77587 SENSE 155 352 CGPG8136 77933 SENSE 156 353 CGPG8152 77619 SENSE 157 354 CGPG8166 77621 SENSE 158 355 CGPG8377 77629 SENSE 159 356 CGPG8976 77832 SENSE 160 357 CGPG8987 76802 SENSE 161 358 CGPG9013 76829 SENSE 162 359 CGPG9080 76961 SENSE 163 360 CGPG9081 76973 SENSE 164 361 CGPG9130 77150 SENSE 165 362 CGPG9133 77186 SENSE 166 363 CGPG9134 77103 SENSE 167 364 CGPG9137 77139 SENSE 168 365 CGPG9141 77187 SENSE 169 366 CGPG9145 77140 SENSE 170 367 CGPG9147 77164 SENSE 171 368 CGPG9148 77176 SENSE 172 369 CGPG9155 77165 SENSE 173 370 CGPG9163 77166 SENSE 174 371 CGPG9170 77155 SENSE 175 372 CGPG9180 77180 SENSE 176 373 CGPG9183 77121 SENSE 177 374 CGPG9186 77157 SENSE 178 375 CGPG9205 77195 SENSE 179 376 CGPG9207 77124 SENSE 180 377 CGPG9219 77261 SENSE 181 378 CGPG9220 77273 SENSE 182 379 CGPG9230 77203 SENSE 183 380 CGPG9236 77275 SENSE 184 381 CGPG9238 77204 SENSE 185 382 CGPG9259 77266 SENSE 186 383 CGPG9271 77220 SENSE 187 384 CGPG9275 77268 SENSE 188 385 CGPG9278 77209 SENSE 189 386 CGPG9283 77269 SENSE 190 387 CGPG9309 77451 SENSE 191 388 CGPG9311 77452 SENSE 192 389 CGPG9322 77430 SENSE 193 390 CGPG9335 77432 SENSE 194 391 CGPG9341 77433 SENSE 195 392 CGPG9344 77444 SENSE 196 393 CGPG9345 77409 SENSE 197 394 CGPG976 12313 ANTI- SENSE 10 207 CGPG1828 16322 SENSE

Recombinant DNA

DNA for use in the present invention to improve traits in plants have a nucleotide sequence of SEQ ID NO:1 through SEQ ID NO:197, as well as the homologs of such DNA molecules. A subset of the DNA for gene suppression aspects of the invention includes fragments of the disclosed full polynucleotides consisting of oligonucleotides of 21 or more consecutive nucleotides. Oligonucleotides the larger molecules having a sequence selected from the group consisting of SEQ ID NO:1 through SEQ ID NO:197 are useful as probes and primers for detection of the polynucleotides used in the invention. Also useful in this invention are variants of the DNA. Such variants may be naturally occurring, including DNA from homologous genes from the same or a different species, or may be non-natural variants, for example DNA synthesized using chemical synthesis methods, or generated using recombinant DNA techniques. Degeneracy of the genetic code provides the possibility to substitute at least one base of the protein encoding sequence of a gene with a different base without causing the amino acid sequence of the polypeptide produced from the gene to be changed. Hence, a DNA useful in the present invention may have any base sequence that has been changed from the sequences provided herein by substitution in accordance with degeneracy of the genetic code.

Homologs of the genes providing DNA demonstrated as useful in improving traits in model plants disclosed herein will generally have significant identity with the DNA disclosed herein. DNA is substantially identical to a reference DNA if, when the sequences of the polynucleotides are optimally aligned there is about 60% nucleotide equivalence; more preferably 70%; more preferably 80% equivalence; more preferably 85% equivalence; more preferably 90%; more preferably 95%; and/or more preferably 98% or 99% equivalence over a comparison window. A comparison window is preferably at least 50-100 nucleotides, and more preferably is the entire length of the polynucleotide provided herein. Optimal alignment of sequences for aligning a comparison window may be conducted by algorithms; preferably by computerized implementations of these algorithms (for example, the Wisconsin Genetics Software Package Release 7.0-10.0, Genetics Computer Group, 575 Science Dr., Madison, Wis.). The reference polynucleotide may be a full-length molecule or a portion of a longer molecule. Preferentially, the window of comparison for determining polynucleotide identity of protein encoding sequences is the entire coding region.

Proteins useful for imparting improved traits are entire proteins or at least a sufficient portion of the entire protein to impart the relevant biological activity of the protein. Proteins useful for generation of transgenic plants having improved traits include the proteins with an amino acid sequence provided herein as SEQ ID NO: 198 through SEQ ID NO: 394, as well as homologs of such proteins.

Homologs of the proteins useful in the invention are identified by comparison of the amino acid sequence of the protein to amino acid sequences of proteins from the same or different plant sources, e.g., manually or by using known homology-based search algorithms such as those commonly known and referred to as BLAST, FASTA, and Smith-Waterman. As used herein, a homolog is a protein from the same or a different organism that performs the same biological function as the polypeptide to which it is compared. An orthologous relation between two organisms is not necessarily manifest as a one-to-one correspondence between two genes, because a gene can be duplicated or deleted after organism phylogenetic separation, such as speciation. For a given protein, there may be no ortholog or more than one ortholog. Other complicating factors include alternatively spliced transcripts from the same gene, limited gene identification, redundant copies of the same gene with different sequence lengths or corrected sequence. A local sequence alignment program, e.g., BLAST, can be used to search a database of sequences to find similar sequences, and the summary Expectation value (E-value) used to measure the sequence base similarity. As a protein hit with the best E-value for a particular organism may not necessarily be an ortholog or the only ortholog, a reciprocal BLAST search is used in the present invention to filter hit sequences with significant E-values for ortholog identification. The reciprocal BLAST entails search of the significant hits against a database of amino acid sequences from the base organism that are similar to the sequence of the query protein. A hit is a likely ortholog, when the reciprocal BLAST's best hit is the query protein itself or a protein encoded by a duplicated gene after speciation. Thus, homolog is used herein to describe proteins that are assumed to have functional similarity by inference from sequence base similarity. The relationship of homologs with amino acid sequences of SEQ ID NO: 395 to SEQ ID NO: 19,938 to the proteins with amino acid sequences of SEQ ID NO: to 198 to SEQ ID NO: 394 are found in the listing of Table 2.

Other functional homolog proteins differ in one or more amino acids from those of a trait-improving protein disclosed herein as the result of one or more of the well-known conservative amino acid substitutions, e.g., valine is a conservative substitute for alanine and threonine is a conservative substitute for serine. Conservative substitutions for an amino acid within the native sequence can be selected from other members of a class to which the naturally occurring amino acid belongs. Representative amino acids within these various classes include, but are not limited to: (1) acidic (negatively charged) amino acids such as aspartic acid and glutamic acid; (2) basic (positively charged) amino acids such as arginine, histidine, and lysine; (3) neutral polar amino acids such as glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; and (4) neutral nonpolar (hydrophobic) amino acids such as alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine. Conserved substitutes for an amino acid within a native amino acid sequence can be selected from other members of the group to which the naturally occurring amino acid belongs. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine. Naturally conservative amino acids substitution groups are: valine-leucine, valine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, aspartic acid-glutamic acid, and asparagine-glutamine. A further aspect of the invention comprises proteins that differ in one or more amino acids from those of a described protein sequence as the result of deletion or insertion of one or more amino acids in a native sequence.

Homologs of the trait-improving proteins provided herein will generally demonstrate significant sequence identity. Of particular interest are proteins having at least 50% sequence identity, more preferably at least about 70% sequence identity or higher, e.g., at least about 80% sequence identity with an amino acid sequence of SEQ ID NO:198 through SEQ ID NO: 394. Of course useful proteins also include those with higher identity, e.g., 90% to 99% identity. Identity of protein homologs is determined by optimally aligning the amino acid sequence of a putative protein homolog with a defined amino acid sequence and by calculating the percentage of identical and conservatively substituted amino acids over the window of comparison. The window of comparison for determining identity can be the entire amino acid sequence disclosed herein, e.g., the full sequence of any of SEQ ID NO: 198 through SEQ ID NO: 394.

Genes that are homologous to each other can be grouped into families and included in multiple sequence alignments. Then a consensus sequence for each group can be derived. This analysis enables the derivation of conserved and class-(family) specific residues or motifs that are functionally important. These conserved residues and motifs can be further validated with 3D protein structure if available. The consensus sequence can be used to define the full scope of the invention, e.g., to identify proteins with a homolog relationship. Thus, the present invention contemplates that protein homologs include proteins with an amino acid sequence that has at least 90% identity to such a consensus amino acid sequence sequences.

Promoters

Numerous promoters that are active in plant cells have been described in the literature. These include promoters present in plant genomes as well as promoters from other sources, including nopaline synthase (NOS) promoter and octopine synthase (OCS) promoters carried on tumor-inducing plasmids of Agrobacterium tumefaciens, caulimovirus promoters such as the cauliflower mosaic virus or Figwort mosaic virus promoters. For instance, see U.S. Pat. Nos. 5,858,742 and 5,322,938 which disclose versions of the constitutive promoter derived from cauliflower mosaic virus (CaMV35S), U.S. Pat. No. 5,378,619 which discloses a Figwort Mosaic Virus (FMV) 35S promoter, U.S. Pat. No. 6,437,217 which discloses a maize RS81 promoter, U.S. Pat. No. 5,641,876 which discloses a rice actin promoter, U.S. Pat. No. 6,426,446 which discloses a maize RS324 promoter, U.S. Pat. No. 6,429,362 which discloses a maize PR-1 promoter, U.S. Pat. No. 6,232,526 which discloses a maize A3 promoter, U.S. Pat. No. 6,177,611 which discloses constitutive maize promoters, U.S. Pat. No. 6,433,252 which discloses a maize L3 oleosin promoter, U.S. Pat. No. 6,429,357 which discloses a rice actin 2 promoter and intron, U.S. Pat. No. 5,837,848 which discloses a root specific promoter, U.S. Pat. No. 6,084,089 which discloses cold inducible promoters, U.S. Pat. No. 6,294,714 which discloses light inducible promoters, U.S. Pat. No. 6,140,078 which discloses salt inducible promoters, U.S. Pat. No. 6,252,138 which discloses pathogen inducible promoters, U.S. Pat. No. 6,175,060 which discloses phosphorus deficiency inducible promoters, U.S. Patent Application Publication 2002/0192813A1 which discloses 5′, 3′ and intron elements useful in the design of effective plant expression vectors, U.S. patent application Ser. No. 09/078,972 which discloses a coixin promoter, U.S. patent application Ser. No. 09/757,089 which discloses a maize chloroplast aldolase promoter, and U.S. patent application Ser. No. 10/739,565 which discloses water-deficit inducible promoters, all of which are incorporated herein by reference. These and numerous other promoters that function in plant cells are known to those skilled in the art and available for use in recombinant polynucleotides of the present invention to provide for expression of desired genes in transgenic plant cells.

Furthermore, the promoters can include multiple “enhancer sequences” to assist in elevating gene expression. Such enhancers are known in the art. By including an enhancer sequence with such constructs, the expression of the selected protein may be enhanced. These enhancers often are found 5′ to the start of transcription in a promoter that functions in eukaryotic cells, but can often be inserted in the forward or reverse orientation 5′ or 3′ to the coding sequence. In some instances, these 5′ enhancing elements are introns. Deemed to be particularly useful as enhancers are the 5′ introns of the rice actin 1 and rice actin 2 genes. Examples of other enhancers that can be used in accordance with the invention include elements from the CaMV 35S promoter, octopine synthase genes, the maize alcohol dehydrogenase gene, the maize shrunken 1 gene and promoters from non-plant eukaryotes.

In some aspects of the invention it is preferred that the promoter element in the DNA construct be capable of causing sufficient expression to result in the production of an effective amount of a polypeptide in water deficit conditions. Such promoters can be identified and isolated from the regulatory region of plant genes that are over expressed in water deficit conditions. Specific water-deficit-inducible promoters for use in this invention are derived from the 5′ regulatory region of genes identified as a heat shock protein 17.5 gene (HSP17.5), an HV A22 gene (HVA22), a Rab17 gene and a cinnamic acid 4-hydroxylase (CA4H) gene (CA4H) of Zea maize. Such water-deficit-inducible promoters are disclosed in U.S. application Ser. No. 10/739,565, incorporated herein by reference.

In some aspects of the invention, sufficient expression in plant seed tissues is desired to effect improvements in seed composition. Exemplary promoters for use for seed composition modification include promoters from seed genes such as napin (U.S. Pat. No. 5,420,034), maize L3 oleosin (U.S. Pat. No. 6,433,252), zein Z27 (Russell et al., (1997) Transgenic Res. 6(2):157-166), globulin 1 (Belanger et al., (1991) Genetics 129:863-872), glutelin 1 (Russell (1997) supra), and peroxiredoxin antioxidant (Per1) (Stacy et al., (1996) Plant Mol. Biol. 31(6):1205-1216).

In some aspects of the invention, preferential expression in plant green tissues is desired. Promoters of interest for such uses include those from genes such as SSU (Fischhoff, et al., (1992) Plant Mol. Biol. 20:81-93), aldolase and pyruvate orthophosphate dikinase (PPDK) (Taniguchi, et al., (2000) Plant Cell Physiol. 41(1):42-48).

Gene suppression includes any of the well-known methods for suppressing transcription of a gene or the accumulation of the mRNA corresponding to that gene thereby preventing translation of the transcript into protein. Posttranscriptional gene suppression is mediated by transcription of RNA that forms double-stranded RNA (dsRNA) having homology to a gene targeted for suppression. Suppression can also be achieved by insertion mutations created by transposable elements may also prevent gene function. For example, in many dicot plants, transformation with the T-DNA of Agrobacterium may be readily achieved and large numbers of transformants can be rapidly obtained. Also, some species have lines with active transposable elements that can efficiently be used for the generation of large numbers of insertion mutations, while some other species lack such options. Mutant plants produced by Agrobacterium or transposon mutagenesis and having altered expression of a polypeptide of interest can be identified using the polynucleotides of the present invention. For example, a large population of mutated plants may be screened with polynucleotides encoding the polypeptide of interest to detect mutated plants having an insertion in the gene encoding the polypeptide of interest.

Gene Stacking

The present invention also contemplates that the trait-improving recombinant DNA provided herein can be used in combination with other recombinant DNA to create plants with multiple desired traits or a further enhanced trait. The combinations generated can include multiple copies of any one or more of the recombinant DNA constructs. These stacked combinations can be created by any method, including but not limited to cross breeding of transgenic plants, or multiple genetic transformation.

In particular embodiments, the inventors contemplate the use of antibodies, either monoclonal or polyclonal which bind to the proteins disclosed herein. Means for preparing and characterizing antibodies are well known in the art (See, e.g., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; incorporated herein by reference). The methods for generating monoclonal antibodies (mAbs) generally begin along the same lines as those for preparing polyclonal antibodies. Briefly, a polyclonal antibody is prepared by immunizing an animal with an immunogenic composition in accordance with the present invention and collecting antisera from that immunized animal. A wide range of animal species can be used for the production of antisera. Typically the animal used for production of anti-antisera is a rabbit, a mouse, a rat, a hamster, a guinea pig or a goat. Because of the relatively large blood volume of rabbits, a rabbit is a preferred choice for production of polyclonal antibodies.

As is well known in the art, a given composition may vary in its immunogenicity. It is often necessary therefore to boost the host immune system, as may be achieved by coupling a peptide or polypeptide immunogen to a carrier. Exemplary and preferred carriers are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albumins such as ovalbumin, mouse serum albumin or rabbit serum albumin can also be used as carriers. Means for conjugating a polypeptide to a carrier protein are well known in the art and include using glutaraldehyde, m-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimide and bis-biazotized benzidine.

As is also well known in the art, the immunogenicity of a particular immunogen composition can be enhanced by the use of non-specific stimulators of the immune response, known as adjuvants. Exemplary and preferred adjuvants include complete Freund's adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis), incomplete Freund's adjuvants and aluminum hydroxide adjuvant.

The amount of immunogen composition used in the production of polyclonal antibodies varies upon the nature of the immunogen as well as the animal used for immunization. A variety of routes can be used to administer the immunogen (subcutaneous, intramuscular, intradermal, intravenous and intraperitoneal). The production of polyclonal antibodies may be monitored by sampling blood of the immunized animal at various points following immunization. A second, booster, injection may also be given. The process of boosting and titering is repeated until a suitable titer is achieved. When a desired level of immunogenicity is obtained, the immunized animal can be bled and the serum isolated and stored, and/or the animal can be used to generate mAbs.

mAbs may be readily prepared through use of well-known techniques, such as those exemplified in U.S. Pat. No. 4,196,265, incorporated herein by reference. Typically, this technique involves immunizing a suitable animal with a selected immunogen composition, e.g., a purified or partially purified antifungal protein, polypeptide or peptide. The immunizing composition is administered in a manner effective to stimulate antibody producing cells. Rodents such as mice and rats are preferred animals, however, the use of rabbit, sheep, or frog cells is also possible. The use of rats may provide certain advantages (Goding, 1986, pp. 60-61), but mice are preferred, with the BALB/c mouse being most preferred as this is most routinely used and generally gives a higher percentage of stable fusions.

Following immunization, somatic cells with the potential for producing antibodies, specifically B lymphocytes (B cells), are selected for use in the mAb generating protocol. These cells may be obtained from biopsied spleens, tonsils or lymph nodes, or from a peripheral blood sample. Spleen cells and peripheral blood cells are preferred, the former because they are a rich source of antibody-producing cells that are in the dividing plasmablast stage, and the latter because peripheral blood is easily accessible. Often, a panel of animals will have been immunized and the spleen of animal with the highest antibody titer will be removed and the spleen lymphocytes obtained by homogenizing the spleen with a syringe. Typically, a spleen from an immunized mouse contains approximately 5×10⁷ to 2×10⁸ lymphocytes.

The antibody-producing B lymphocytes from the immunized animal are then fused with cells of an immortal myeloma cell, generally one of the same species as the animal that was immunized. Myeloma cell lines suited for use in hybridoma-producing fusion procedures preferably are non-antibody-producing, have high fusion efficiency, and enzyme deficiencies that render them incapable of growing in certain selective media which support the growth of only the desired fused cells (hybridomas).

Any one of a number of myeloma cells may be used, as are known to those of skill in the art (Goding, 1986, pp. 65-66; Campbell, 1984, pp. 75-83). For example, where the immunized animal is a mouse, one may use P3-X63/Ag8, X63-Ag8.653, NS1/1.Ag 4 1, Sp210-Ag14, FO, NSO/U, MPC-11, MPC11-X45-GTG 1.7 and S194/5XXO Bu1; for rats, one may use R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B210; and U-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6 are all useful in connection with human cell fusions.

One preferred murine myeloma cell is the NS-1 myeloma cell line (also termed P3-NS-1-Ag-4-1), which is readily available from the NIGMS Human Genetic Mutant Cell Repository by requesting cell line repository number GM3573. Another mouse myeloma cell line that may be used is the 8-azaguanine-resistant mouse murine myeloma SP2/0 non-producer cell line.

Methods for generating hybrids of antibody-producing spleen or lymph node cells and myeloma cells usually comprise mixing somatic cells with myeloma cells in a 2:1 ratio, though the ratio may vary from about 20:1 to about 1:1, respectively, in the presence of an agent or agents (chemical or electrical) that promote the fusion of cell membranes. Fusion methods using Spend virus have been described (Kohler and Milstein, 1975; 1976), and those using polyethylene glycol (PEG), such as 37% (v/v) PEG, (Gefter et al., 1977). The use of electrically induced fusion methods is also appropriate (Goding, 1986, pp. 71-74).

Fusion procedures usually produce viable hybrids at low frequencies, about 1×10⁻⁶ to 1×10⁻⁸. However, this does not pose a problem, as the viable, fused hybrids are differentiated from the parental, unfused cells (particularly the unfused myeloma cells that would normally continue to divide indefinitely) by culturing in a selective medium. The selective medium is generally one that contains an agent that blocks the de novo synthesis of nucleotides in the tissue culture media. Exemplary and preferred agents are aminopterin, methotrexate, and azaserine. Aminopterin and methotrexate block de novo synthesis of both purines and pyrimidines, whereas azasenne blocks only purine synthesis. Where aminopterin or methotrexate is used, the media is supplemented with hypoxanthine and thymidine as a source of nucleotides (HAT medium). Where azaserine is used, the media is supplemented with hypoxanthine.

The preferred selection medium is HAT. Only cells capable of operating nucleotide salvage pathways are able to survive in HAT medium. The myeloma cells are defective in key enzymes of the salvage pathway, e.g., hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive. The B-cells can operate this pathway, but they have a limited life span in culture and generally die within about two weeks. Therefore, the only cells that can survive in the selective media are those hybrids formed from myeloma and B-cells.

This culturing provides a population of hybridomas from which specific hybridomas are selected. Typically, selection of hybridomas is performed by culturing the cells by single-clone dilution in microtiter plates, followed by testing the individual clonal supernatants (after about two to three weeks) for the desired reactivity. The assay should be sensitive, simple and rapid, such as radioimmunoassays, enzyme immunoassays, cytotoxicity assays, plaque assays, dot immunobinding assays, and the like.

The selected hybridomas would then be serially diluted and cloned into individual antibody-producing cell lines, which clones can then be propagated indefinitely to provide mAbs. The cell lines may be exploited for mAb production in two basic ways. A sample of the hybridoma can be injected (often into the peritoneal cavity) into a histocompatible animal of the type that was used to provide the somatic and myeloma cells for the original fusion. The injected animal develops tumors secreting the specific monoclonal antibody produced by the fused cell hybrid. The body fluids of the animal, such as serum or ascites fluid, can then be tapped to provide mAbs in high concentration. The individual cell lines could also be cultured in vitro, where the mAbs are naturally secreted into the culture medium from which they can be readily obtained in high concentrations. mAbs produced by either means may be further purified, if desired, using filtration, centrifugation and various chromatographic methods such as HPLC or affinity chromatography.

Transformation Methods

Numerous methods for producing plant cell nuclei with recombinant DNA are known in the art and may be used in the present invention. Two commonly used methods for plant transformation are Agrobacterium-mediated transformation and microprojectile bombardment. Microprojectile bombardment methods are illustrated in U.S. Pat. Nos. 5,015,580 (soybean); 5,550,318 (corn); 5,538,880 (corn); 5,914,451 (soybean); 6,160,208 (corn); 6,399,861 (corn) and 6,153,812 (wheat) and Agrobacterium-mediated transformation is described in U.S. Pat. Nos. 5,159,135 (cotton); 5,824,877 (soybean); 5,591,616 (corn); and 6,384,301 (soybean), all of which are incorporated herein by reference. For Agrobacterium tumefaciens based plant transformation system, additional elements present on transformation constructs will include T-DNA left and right border sequences to facilitate incorporation of the recombinant polynucleotide into the plant genome.

In general it is preferred to introduce heterologous DNA randomly, i.e., at a non-specific location, in the genome of a target plant line. In special cases it may be useful to target heterologous DNA insertion in order to achieve site-specific integration, e.g., to replace an existing gene in the genome, to use an existing promoter in the plant genome, or to insert a recombinant polynucleotide at a predetermined site known to be active for gene expression. Several site specific recombination systems exist which are known to function implants include cre-lox as disclosed in U.S. Pat. No. 4,959,317 and FLP-FRT as disclosed in U.S. Pat. No. 5,527,695, both incorporated herein by reference.

Transformation methods of this invention are preferably practiced in tissue culture on media and in a controlled environment. “Media” refers to the numerous nutrient mixtures that are used to grow cells in vitro, that is, outside of the intact living organism. Recipient cell targets include, but are not limited to, meristem cells, callus, immature embryos and gametic cells such as microspores, pollen, sperm and egg cells. It is contemplated that any cell from which a fertile plant may be regenerated is useful as a recipient cell. Callus may be initiated from tissue sources including, but not limited to, immature embryos, seedling apical meristems, microspores and the like. Cells capable of proliferating as callus are also recipient cells for genetic transformation. Practical transformation methods and materials for making transgenic plants of this invention, e.g., various media and recipient target cells, transformation of immature embryos and subsequent regeneration of fertile transgenic plants are disclosed in U.S. Pat. Nos. 6,194,636 and 6,232,526 and U.S. patent application Ser. No. 09/757,089, which are incorporated herein by reference.

In practice DNA is introduced into only a small percentage of target cell nuclei in any one experiment. Marker genes are used to provide an efficient system for identification of those cells with nuclei that are stably transformed by receiving and integrating a transgenic DNA construct into their genomes. Preferred marker genes provide selective markers that confer resistance to a selective agent, such as an antibiotic or herbicide. Potentially transformed cells with a nucleus of the invention are exposed to the selective agent. In the population of surviving cells will be those cells where, generally, the resistance-conferring gene has been integrated and expressed at sufficient levels to permit cell survival. Cells may be tested further to confirm stable integration of the exogenous DNA in the nucleus. Useful selective marker genes include those conferring resistance to antibiotics such as kanamycin (nptII), hygromycin B (aph IV) and gentamycin (aac3 and aacC4) or resistance to herbicides such as glufosinate (bar or pat) and glyphosate (EPSPS). Examples of such selectable are illustrated in U.S. Pat. Nos. 5,550,318; 5,633,435; 5,780,708 and 6,118,047, all of which are incorporated herein by reference. Screenable markers which provide an ability to visually identify transformants can also be employed, e.g., a gene expressing a colored or fluorescent protein such as a luciferase or green fluorescent protein (GFP) or a gene expressing a beta-glucuronidase or uidA gene (GUS) for which various chromogenic substrates are known. It is also contemplated that combinations of screenable and selectable markers will be useful for identification of transformed cells. See PCT publication WO 99/61129 which discloses use of a gene fusion between a selectable marker gene and a screenable marker gene, e.g., an NPTII gene and a GFP gene.

Cells that survive exposure to the selective agent, or cells that have been scored positive in a screening assay, may be cultured in regeneration media and allowed to mature into plants. Developing plantlets can be transferred to soil less plant growth mix, and hardened off, e.g., in an environmentally controlled chamber at about 85% relative humidity, 600 ppm CO₂, and 25-250 microeinsteins m⁻² s⁻¹ of light, prior to transfer to a greenhouse or growth chamber for maturation. Plants are preferably matured either in a growth chamber or greenhouse. Plants are regenerated from about 6 wk to 10 months after a transformant is identified, depending on the initial tissue. During regeneration, cells are grown to plants on solid media at about 19 to 28° C. After regenerating plants have reached the stage of shoot and root development, they may be transferred to a greenhouse for further growth and testing. Plants may be pollinated using conventional plant breeding methods known to those of skill in the art and seed produced.

Progeny may be recovered from transformed plants and tested for expression of the exogenous recombinant polynucleotide. Useful assays include, for example, “molecular biological” assays, such as Southern and Northern blotting and PCR; “biochemical” assays, such as detecting the presence of RNA, e.g., double stranded RNA, or a protein product, e.g., by immunological means (ELISAs and Western blots) or by enzymatic function; plant part assays, such as leaf or root assays; and also, by analyzing the phenotype of the whole regenerated plant.

Discovery of Trait-improving Recombinant DNA

To identify nuclei with recombinant DNA that confer improved traits to plants, Arabidopsis thaliana was transformed with a candidate recombinant DNA construct and screened for an improved trait.

Arabidopsis thaliana is used a model for genetics and metabolism in plants. Arabidopsis has a small genome, and well-documented studies are available. It is easy to grow in large numbers and mutants defining important genetically controlled mechanisms are either available, or can readily be obtained. Various methods to introduce and express isolated homologous genes are available (see Koncz, e.g., Methods in Arabidopsis Research e.g., (1992), World Scientific, New Jersey, New Jersey, in “Preface”).

A two-step screening process was employed which comprised two passes of trait characterization to ensure that the trait modification was dependent on expression of the recombinant DNA, but not due to the chromosomal location of the integration of the transgene. Twelve independent transgenic lines for each recombinant DNA construct were established and assayed for the transgene expression levels. Five transgenic lines with high transgene expression levels were used in the first pass screen to evaluate the transgene's function in T2 transgenic plants. Subsequently, three transgenic events, which had been shown to have one or more improved traits, were further evaluated in the second pass screen to confirm the transgene's ability to impart an improved trait. The following Table 3 summarizes the improved traits that have been confirmed as provided by a recombinant DNA construct.

In particular, Table 3 reports:

“PEP SEQ ID” which is the amino acid sequence of the protein cognate to the DNA in the recombinant DNA construct corresponding to a protein sequence of a SEQ ID NO. in the Sequence Listing.

“construct_id” is an arbitrary name for the recombinant DNA describe more particularly in Table 1.

“annotation” refers to a description of the top hit protein obtained from an amino acid sequence query of each PEP SEQ ID NO to GenBank database of the National Center for Biotechnology Information (ncbi). More particularly, “gi” is the GenBank ID number for the top BLAST hit.

“description” refers to the description of the top BLAST hit.

“e-value” provides the expectation value for the BLAST hit.

“% id” refers to the percentage of identically matched amino acid residues along the length of the portion of the sequences which is aligned by BLAST between the sequence of interest provided herein and the hit sequence in GenBank.

“traits” identify by two letter codes the confirmed improvement in a transgenic plant provided by the recombinant DNA. The codes for improved traits are:

“CK” which indicates cold tolerance improvement identified under a cold shock tolerance screen;

“CS” which indicates cold tolerance improvement identified by a cold germination tolerance screen;

“DS” which indicates drought tolerance improvement identified by a soil drought stress tolerance screen;

“PEG” which indicates osmotic stress tolerance improvement identified by a PEG induced osmotic stress tolerance screen;

“HS” which indicates heat stress tolerance improvement identified by a heat stress tolerance screen;

“SS” which indicates high salinity stress tolerance improvement identified by a salt stress tolerance screen;

“LN” which indicates nitrogen use efficiency improvement identified by a limited nitrogen tolerance screen;

“LL” which indicates attenuated shade avoidance response identified by a shade tolerance screen under a low light condition;

“PP” which indicates improved growth and development at early stages identified by an early plant growth and development screen;

“SP” which indicates improved growth and development at late stages identified by a late plant growth and development screen provided herein.

TABLE 3 PEP Annotation SEQ Construct e % ID ID value id description trait 198 11029 1.00E−170 85 gb|AAD02882.1|gamma-tocopherol DS LN methyltransferase [Arabidopsis thaliana] 199 12223 / / / CK PEG 200 10422 1.00E−133 100 gb|AAB47766.1|SNF5 homolog BSH LN [Arabidopsis thaliana] 201 13485 4.00E−39 55 emb|CAB79347.1|hypothetical LN protein [Arabidopsis thaliana] 202 13235 7.00E−76 100 emb|CAB78153.1|putative protein LN [Arabidopsis thaliana] 203 13411 3.00E−29 100 ref|NP_179638.1|unknown protein LN CK [Arabidopsis thaliana] 204 73944 0 95 ref|NP_189578.1|phosphorylase/ CS transferase, transferring glycosyl groups [Arabidopsis thaliana] 205 13846 0 91 gb|AAN60291.1|unknown LN [Arabidopsis thaliana] 206 12602 0 100 ref|NP_565974.1|PP2A-4; hydrolase/ LN protein phosphatase type 2A/protein serine/threonine phosphatase [Arabidopsis thaliana] 207 74065 0 94 ref|NP_181390.1|DNA binding/ CS DS transcription factor [Arabidopsis thaliana] gb|AAN13033.1|putative elongation 208 72783 0 83 ref|NP_564690.1|unknown protein CK PEG HS [Arabidopsis thaliana] 209 17210 1.00E−92 100 ref|NP_563622.1|unknown protein CS CK PEG [Arabidopsis thaliana] 210 72724 1.00E−128 90 ref|NP_190525.1|protein translocase/ LL protein transporter [Arabidopsis thaliana 211 17805 1.00E−172 80 ref|NP_563761.1|unknown protein LN [Arabidopsis thaliana] 212 16610 0 100 gb|AAL44550.1|fructose bisphosphate LN HS aldolase [Agrobacterium tumefaciens str. C58] 213 76602 0 78 ref|NP_566515.1|signal transducer CS HS [Arabidopsis thaliana] 214 18456 1.00E−135 81 ref|NP_176820.1|DNA binding/ CK SS PEG transcription factor [Arabidopsis thaliana] 215 18414 1.00E−169 70 ref|NP_195583.1|translation initiation CK factor [Arabidopsis thaliana] 216 71538 1.00E−137 90 emb|CAE85115.1|synaptotagmin SP [Arabidopsis thaliana] 217 76532 0 99 ref|NP_564775.1|ATP binding/ CK CS carbohydrate binding/kinase/protein kinase/protein serine/threonine kinase/protein-tyrosine kinase/sugar binding [Arabidopsis thaliana] 218 18231 1.00E−142 100 emb|CAC83762.1|allene oxide CK LN cyclase [Arabidopsis thaliana] 219 18232 6.00E−85 89 ref|NP_191628.1|unknown protein CS [Arabidopsis thaliana] 220 18256 1.00E−102 100 dbj|BAD44508.1|putative zinc finger SS CK HS protein (PMZ) [Arabidopsis thaliana] 221 18258 1.00E−175 83 emb|CAA11525.1|transcription factor SP IIA large subunit [Arabidopsis thaliana] 222 19193 1.00E−103 100 ref|NP_564682.1|lipid binding SP HS [Arabidopsis thaliana] dbj|BAE73268.1|xylogen like protein 12 [Arabidopsis thaliana] 223 71301 0 84 ref|NP_568393.1|nucleic acid binding LN [Arabidopsis thaliana] 224 18354 0 96 ref|NP_567568.1|ATP binding/ LN kinase/protein kinase/protein serine/threonine kinase/protein- tyrosine kinase [Arabidopsis thaliana] 225 77334 1.00E−117 100 ref|NP_178399.1|RNS1 LL (RIBONUCLEASE 1); endoribonuclease [Arabidopsis thaliana] 226 19767 1.00E−180 95 gb|AAO69665.1|serine threonine CS DS protein phosphatase [Phaseolus acutifolius] 227 19774 1.00E−100 57 ref|NP_974271.1|unknown protein CS PP [Arabidopsis thaliana] 228 70992 1.00E−108 65 gb|ABE87200.1|hypothetical protein CS PEG MtrDRAFT_AC151668g4v1 [Medicago truncatula] 229 19956 1.00E−146 100 gb|AAF64040.1|14-3-3-like protein PP [Glycine max] 230 70948 3.00E−59 45 ref|NP_201297.1|CIP8 (COP1- PP HS INTERACTING PROTEIN 8); protein binding/zinc ion binding [Arabidopsis thaliana] 231 70201 0 68 ref|NP_567072.1|ATP binding/ CS SS kinase/protein kinase/protein serine/threonine kinase/protein- tyrosine kinase [Arabidopsis thaliana] 232 19973 0 96 gb|AAB04057.1|S-adenosyl-L- PP SS methionine:delta24-sterol-C- methyltransferase 233 70987 0 93 gb|AAM83095.1|SOS2-like protein CK CS HS SS PEG kinase [Glycine max] 234 70950 1.00E−116 77 dbj|BAD38167.1|putative leucine PP PEG zipper protein [Oryza sativa (japonica cultivar-group)] 235 70962 1.00E−179 78 gb|ABE93200.1|conserved PP HS hypothetical protein [Medicago truncatula] 236 70915 0 76 dbj|BAE71210.1|hypothetical protein CS PP [Trifolium pratense] dbj|BAE71208.1| hypothetical protein [Trifolium pratense] 237 19947 1.00E−116 65 gb|AAS38575.1|short-chain CS DS SS dehydrogenase Tic32 [Pisum sativum] 238 70969 1.00E−144 76 ref|NP_174321.2|unknown protein SS [Arabidopsis thaliana] 239 70985 0 69 dbj|BAB08296.1|unnamed protein CS HS PEG product [Arabidopsis thaliana] sp|Q9FMT4|Y5417_ARATH Protein At5g14170 240 70971 1.00E−92 63 gb|ABE80664.1|GNS1/SUR4 SS membrane protein [Medicago truncatula] gb|ABE78502.1| GNS1/SUR4 membrane protein [Medicago truncatula] 241 70963 1.00E−96 56 ref|NP_177178.1|CYCD1; 1; cyclin- CS PP HS dependent protein kinase regulator [Arabidopsis thaliana] 242 70994 2.00E−33 44 ref|NP_172962.1|RHA2A; protein PP DS HS SS binding/ubiquitin-protein ligase/zinc ion binding [Arabidopsis thaliana] 243 70956 0 85 gb|AAO23063.1|ent-kaurenoic acid CS SS HS oxidase [Pisum sativum] 244 70995 1.00E−132 66 dbj|BAD73789.1|putative CK CS PP LL PEG uncharacterized hypothalamus protein HT010 [Oryza sativa (japonica cultivar-group)] 245 73306 0 93 ref|NP_201243.1|phosphoric ester SS PEG hydrolase [Arabidopsis thaliana] 246 70657 9.00E−31 73 emb|CAB78150.1|probable wound- SP LL induced protein [Arabidopsis thaliana] 247 70660 2.00E−32 79 ref|NP_568217.1|transcription SP regulator [Arabidopsis thaliana] 248 74536 0 91 ref|NP_001031061.1|PGDH; CS phosphoglycerate dehydrogenase [Arabidopsis thaliana]] 249 71328 0 99 gb|AAD46022.1|Strong simlarity to PP gb|286426 F10M6.190 cytochrome p450 homolog from Arabidopsis thaliana BAC 250 71329 0 94 ref|NP_177477.1|heme binding/iron LN ion binding/monooxygenase/oxygen binding [Arabidopsis thaliana] 251 70755 1.00E−142 100 ref|NP_188925.1|unknown protein LL [Arabidopsis thaliana] 252 70684 3.00E−53 100 emb|CAB80194.1|putative protein CS LN HS PP [Arabidopsis thaliana] 253 73330 1.00E−158 72 emb|CAB96855.1|putative protein CK CS SS [Arabidopsis thaliana] 254 76202 3.00E−83 67 emb|CAA16586.1|putative protein LL [Arabidopsis thaliana] 255 72807 0 100 ref|NP_173478.2|nucleotide binding PEG CK HS SS [Arabidopsis thaliana] 256 72811 1.00E−176 100 ref|NP_565782.1|nucleotide binding CK SS PEG [Arabidopsis thaliana] 257 73235 0 89 ref|NP_564867.1|pepsin A SS HS [Arabidopsis thaliana] 258 72813 2.00E−79 93 ref|NP_850506.1|unknown protein LL LN [Arabidopsis thaliana] 259 72825 1.00E−154 95 ref|NP_563635.1|oxidoreductase CK PEG HS [Arabidopsis thaliana] 260 73628 1.00E−163 92 ref|NP_849428.1|oxidoreductase CS DS LL HS PEG [Arabidopsis thaliana] 261 76105 0 93 emb|CAB79123.1|receptor kinase-like DS protein [Arabidopsis thaliana] 262 73242 0 100 ref|NP_567292.1|unknown protein SP PEG [Arabidopsis thaliana] 263 74217 0 100 emb|CAB93726.1|cytochrome P450- DS CK like protein [Arabidopsis thaliana] 264 73735 0 97 gb|AAU94404.1|At3g48520 CK [Arabidopsis thaliana] 265 73256 1.00E−114 77 ref|NP_566329.1|unknown protein CS PP PEG [Arabidopsis thaliana] gb|AAL06975.1| 266 73260 4.00E−37 100 ref|NP_566488.1|unknown protein CS [Arabidopsis thaliana] 267 75822 1.00E−90 88 emb|CAB63015.1|putative protein LL [Arabidopsis thaliana] 268 72001 0 96 ref|NP_200045.1|CYP96A4; heme CS PP HS binding/iron ion binding/ monooxygenase/oxygen binding [Arabidopsis thaliana] 269 72056 0 92 ref|NP_010186.1|Essential, non- SP ATPase regulatory subunit of the 26S proteasome lid required for the assembly and activity of the 26S proteasome; 270 77308 0 100 ref|NP_178642.1|SCPL38; serine PEG carboxypeptidase [Arabidopsis thaliana] 271 73766 1.00E−163 100 emb|CAB62363.1|MTN3-like protein LL [Arabidopsis thaliana] 272 72774 1.00E−139 95 ref|NP_011052.1|Constituent of 66S LL CK HS pre-ribosomal particles, involved in 60S ribosomal subunit biogenesis; 273 72788 0 81 ref|NP_010007.1|General repressor of CK CS SS transcription, forms complex with Cyc8p, involved in the establishment of repressive chromatin structure through interactions with histones H3 and H4, appears to enhance expression of some genes 274 72753 1.00E−105 85 ref|NP_013125.1|One of four subunits CS PP HS PEG of the endosomal sorting complex required for transport III (ESCRT- III); 275 72709 0 94 ref|NP_011827.1|Low affinity LL methionine permease, similar to Mup1p; 276 73954 0 98 ref|NP_200954.1|MIM; ATP binding HS [Arabidopsis thaliana] 277 73137 1.00E−110 94 gb|AAD32800.1|putative thioredoxin CS H [Arabidopsis thaliana] 278 73161 1.00E−36 65 emb|CAB85993.1|putative protein CS PEG [Arabidopsis thaliana] 279 73057 1.00E−133 95 ref|NP_012770.1|Tetrameric SP phosphoglycerate mutase, mediates the conversion of 3-phosphoglycerate to 2-phosphoglycerate during glycolysis and the reverse reaction during gluconeogenesis; 280 73127 0 99 dbj|BAB05414.1|aspartate SS HS aminotransferase [Bacillus halodurans C-125] ref|NP_242561.1|aspartate aminotransferase [Bacillus halodurans C-125] 281 73033 0 96 dbj|BAB07276.1|2,3- CK bisphosphoglycerate-independent phosphoglycerate mutase [Bacillus halodurans C-125] 282 73105 0 99 emb|CAB13630.1|glutamine PP PEG synthetase [Bacillus subtilis subsp. subtilis str. 168] 283 73141 0 95 ref|NP_414696.1|glutamate-1- CS semialdehyde aminotransferase [Escherichia coli K12] 284 73165 0 99 ref|NP_288110.1|pyruvate kinase DS PEG [Escherichia coli O157:H7 EDL933] 285 73155 1.00E−173 92 emb|CAC47346.1|PROBABLE CS FRUCTOSE-BISPHOSPHATE ALDOLASE CLASS I PROTEIN [Sinorhizobium meliloti]] 286 73026 1.00E−116 87 emb|CAG77169.1|triosephosphate PEG isomerase [Erwinia carotovora subsp. atroseptica SCRI1043] 287 73120 8.00E−82 100 ref|NP_441918.1|nucleoside CS SS HS diphosphate kinase [Synechocystis sp. PCC 6803] 288 73133 2.00E−63 90 gb|ABA76342.1|Nucleoside LL diphosphate kinase [Pseudomonas fluorescens PfO-1] 289 73134 9.00E−98 81 emb|CAE15979.1|ribose 5-phosphate SS CK isomerase A (phosphoriboisomerase A) [Photorhabdus luminescens subsp. laumondii TTO1] 290 73123 0 95 ref|NP_180412.2|nucleic acid binding CS PEG [Arabidopsis thaliana] 291 73981 0 93 ref|NP_014797.1|hypothetical protein; DS PEG Slp1p [Saccharomyces cerevisiae] 292 73136 0 91 ref|NP_012454.1|Nuclear actin- DS PP related protein involved in chromatin remodeling 293 73172 0 100 ref|NP_014952.1|Protein involved in CK PEG HS ER-to-Golgi transport; Sly41p [Saccharomyces cerevisiae] 294 73020 0 93 ref|NP_014993.1|Proline permease, LL required for high-affinity transport of proline 295 72946 0 95 ref|NP_012534.1|Vacuolar PEG LL transporter, imports large neutral amino acids into the vacuole 296 74746 0 93 ref|NP_566298.1|ATP binding/ CK kinase/protein kinase/protein serine/threonine kinase/protein- tyrosine kinase [Arabidopsis thaliana] 297 77312 0 90 ref|NP_178463.1|CDC48B; ATP PP SS HS binding/ATP-dependent peptidase/ ATPase/nucleoside-triphosphatase/ nucleotide binding/serine-type endopeptidase [Arabidopsis thaliana] 298 75237 0 100 ref|NP_173390.1|kinase [Arabidopsis LN thaliana] gb|AAF98405.1|Unknown protein [Arabidopsis thaliana] 299 75240 0 100 emb|CAB69854.1|putative protein CK [Arabidopsis thaliana] 300 74349 1.00E−148 79 ref|NP_565187.1|transcription LN regulator [Arabidopsis thaliana] 301 76422 5.00E−44 100 ref|NP_171781.1|unknown protein LN [Arabidopsis thaliana] 302 70812 1.00E−152 92 ref|NP_850182.1|PUR ALPHA-1; PP PEG nucleic acid binding [Arabidopsis thaliana] 303 77322 1.00E−172 100 gb|AAW38983.1|At5g10750 LN LL [Arabidopsis thaliana] 304 74662 4.00E−89 100 ref|NP_001031939.1|ubiquitin PP HS conjugating enzyme/ubiquitin-like activating enzyme [Arabidopsis thaliana] 305 76527 0 96 emb|CAB51062.1|cell division cycle CS protein 23 homolog [Arabidopsis thaliana] 306 77020 0 98 emb|CAB75781.1|putative transporter CS protein [Arabidopsis thaliana] ref|NP_190154.1|transporter [Arabidopsis thaliana] 307 77609 0 96 gb|AAU95452.1|At5g04420 LL SS [Arabidopsis thaliana] 308 73485 0 100 emb|CAD84238.1|Glyceraldehyde 3- PEG phosphate dehydrogenase 309 73433 1.00E−132 65 ref|ZP_00819153.1|putative alcohol SP LN PP dehydrogenase [Marinobacter aquaeolei VT8] 310 73411 0 99 emb|CAB14878.1|pyruvate kinase CS HS PP PEG [Bacillus subtilis subsp. subtilis str. 168] 311 73568 0 97 emb|CAC41401.1|PROBABLE SP SUCCINATE-SEMIALDEHYDE DEHYDROGENASE [NADP+] PROTEIN [Sinorhizobium meliloti] 312 74688 0 98 emb|CAA16688.1|receptor protein CS PP SS PEG kinase—like protein [Arabidopsis thaliana] 313 74420 1.00E−142 99 emb|CAD85691.1|Phosphoglycerate PP LN SS mutase family [Nitrosomonas europaea ATCC 19718] 314 74435 1.00E−117 100 emb|CAC41549.1|PROBABLE LL PHOSPHOGLYCERATE MUTASE 1 PROTEIN [Sinorhizobium meliloti] 315 74460 0 95 ref|NP_441738.1|fructose-1,6- CK bisphosphatase [Synechocystis sp. PCC 6803] 316 74566 0 93 emb|CAC48499.1|putative trehalose SP PP SS PEG HS synthase protein [Sinorhizobium meliloti 1021] 317 77610 0 94 gb|AAC23406.1|hypothetical protein CS HS [Arabidopsis thaliana] 318 77618 0 100 ref|NP_850453.1|JAR1 PP HS (JASMONATE RESISTANT 1) [Arabidopsis thaliana] 319 77517 7.00E−77 100 emb|CAB75802.1|putative protein CS LL [Arabidopsis thaliana] 320 77518 1.00E−49 100 ref|NP_199600.1|oxidoreductase, SS LL HS acting on NADH or NADPH, quinone or similar compound as acceptor [Arabidopsis thaliana] 321 76460 / / / CS PEG 322 77069 0 85 ref|NP_201196.1|unknown protein PP [Arabidopsis thaliana] 323 76161 1.00E−161 100 ref|NP_851282.1|unknown protein CK [Arabidopsis thaliana] 324 76171 1.00E−178 95 ref|NP_176204.1|oxidoreductase LL [Arabidopsis thaliana] 325 76178 5.00E−60 86 ref|NP_565029.1|unknown protein CS PP HS LL PEG [Arabidopsis thaliana] 326 76467 6.00E−86 89 ref|NP_565671.1|unknown protein LL [Arabidopsis thaliana] 327 77536 1.00E−119 83 gb|AAS99692.1|At1g10020 SP CS DS PP LN [Arabidopsis thaliana] 328 76576 1.00E−145 100 ref|NP_181023.1|FAH1 (FATTY DS ACID HYDROXYLASE 1); catalytic [Arabidopsis thaliana] 329 74862 1.00E−145 100 gb|ABA77057.1|Delta 1-pyrroline-5- PP carboxylate reductase [Pseudomonas fluorescens PfO-1] 330 74863 1.00E−93 100 ref|NP_012420.1|Nucleosome LL PEG CK SS assembly factor, involved in chromatin assembly after DNA replication] 331 74858 5.00E−50 68 gb|AAK14395.1|response regulator LL protein [Dianthus caryophyllus] 332 74933 1.00E−177 95 gb|AAK85899.1|AGR_C_118p LL [Agrobacterium tumefaciens str. C58] 333 75379 1.00E−105 76 dbj|BAD73205.1|unknown protein LN [Oryza sativa (japonica cultivar- group)] 334 77816 4.00E−41 89 gb|ABA98984.1|expressed protein SP PP HS [Oryza sativa (japonica cultivar- group)] 335 75434 3.00E−52 79 ref|XP_472650.1|OSJNBa0027P08.15 LN [Oryza sativa (japonica cultivar- group)] 336 77821 0 71 gb|ABE84883.1|conserved PP SS HS PEG hypothetical protein [Medicago truncatula] 337 75685 0 82 ref|XP_475937.1|unknown protein LL [Oryza sativa (japonica cultivar- group)] 338 75654 0 100 ref|NP_563865.1|unknown protein SP CK HS [Arabidopsis thaliana] 339 75692 1.00E−119 87 dbj|BAD30296.1|peptidyl-prolyl cis- CK LN trans isomerase-like protein [Oryza sativa (japonica cultivar-group)] 340 75657 6.00E−57 67 dbj|BAD38392.1|DNAJ heat shock N- CS LL HS terminal domain-containing protein- like [Oryza sativa (japonica cultivar- group)] 341 75622 1.00E−30 78 dbj|BAD45825.1|unknown protein LN [Oryza sativa (japonica cultivar- group)] 342 77549 1.00E−132 89 emb|CAB80891.1|AT4g00820 PP HS [Arabidopsis thaliana]] 343 77917 0 91 ref|NP_197917.1|EBF2 (EIN3- PP PEG BINDING F BOX PROTEIN 2) [Arabidopsis thaliana] 344 77568 9.00E−99 76 ref|NP_849792.1|nucleic acid binding PP [Arabidopsis thaliana] 345 77570 7.00E−54 100 gb|AAS75309.1|multidomain CK SS cyclophilin type peptidyl-prolyl cis- trans isomerase [Arabidopsis thaliana]] 346 77338 4.00E−24 65 gb|AAM61454.1|unknown PEG [Arabidopsis thaliana] 347 77580 3.00E−45 100 dbj|BAB01457.1|unnamed protein PEG CS HS SS product [Arabidopsis thaliana] 348 77928 4.00E−38 100 ref|NP_196244.1|unknown protein HS [Arabidopsis thaliana] 349 77349 1.00E−81 93 emb|CAC05463.1|putative lipid LL SS transfer protein [Arabidopsis thaliana] 350 77357 1.00E−155 95 ref|NP_175357.1|unknown protein PEG [Arabidopsis thaliana] 351 77587 / / / CS PP HS SS 352 77933 / / / SS 353 77619 3.00E−40 98 dbj|BAB09403.1|unnamed protein CK SS PEG HS product [Arabidopsis thaliana] 354 77621 4.00E−58 74 ref|NP_197632.1|Rac GTPase CK activator [Arabidopsis thaliana] [Arabidopsis thaliana] 355 77629 5.00E−36 100 ref|NP_196372.1|GRP19 [Arabidopsis SS HS thaliana] 356 77832 0 83 ref|NP_917762.1|P0501G01.24 CS SS PEG HS LN [Oryza sativa (japonica cultivar- group)] 357 76802 0 76 ref|NP_181908.1|actin binding CK [Arabidopsis thaliana] gb|AAB64026.1|unknown protein [Arabidopsis thaliana] 358 76829 0 89 ref|NP_909912.1|ferredoxin-NADP+ CK reductase [Oryza sativa] 359 76961 0 65 ref|XP_473189.1|OSJNBa0073E02.11 LL LN [Oryza sativa (japonica cultivar- group)] 360 76973 1.00E−151 76 ref|XP_480055.1|unknown protein LL [Oryza sativa (japonica cultivar- group)] 361 77150 0 76 dbj|BAD61385.1|putative SP HS PEG nucleostemin [Oryza sativa (japonica cultivar-group)] 362 77186 0 85 dbj|BAD27898.1|putative PP pentatricopeptide (PPR) repeat- containing protein [Oryza sativa (japonica cultivar-group)] 363 77103 1.00E−174 66 ref|NP_568580.1|catalytic LL DS [Arabidopsis thaliana] 364 77139 2.00E−93 58 ref|NP_171690.1|PFC1 (PALEFACE CS SS 1) [Arabidopsis thaliana] 365 77187 1.00E−160 83 ref|NP_913437.1|3-methyl-2- CS PP LL PEG oxobutanoate hydroxy-methyl- transferase-like protein [Oryza sativa (japonica cultivar-group)] 366 77140 0 77 dbj|BAD36145.1|membrane protein CK PEG SS PTM1-like [Oryza sativa (japonica cultivar-group)] 367 77164 0 91 gb|ABF93778.1|DNA polymerase PP HS delta small subunit, putative, expressed [Oryza sativa (japonica cultivar-group)] 368 77176 0 76 ref|NP_914476.1|putative LL phytochrome P450 [Oryza sativa (japonica cultivar-group)] 369 77165 1.00E−139 75 ref|NP_914949.1|serine/threonine CK LL LN protein kinase-like protein [Oryza sativa (japonica cultivar-group)] 370 77166 0 85 dbj|BAB75233.1|all3534 [Nostoc sp. CK PP PCC 7120] 371 77155 0 97 emb|CAA35550.1|hycE [Escherichia PP coli] 372 77180 2.00E−62 61 gb|ABA99663.1|expressed protein CS PP HS PEG [Oryza sativa (japonica cultivar- group)] 373 77121 1.00E−166 93 dbj|BAB05404.1|transcriptional CK repressor of the biotin operon [Bacillus halodurans C-125] 374 77157 0 100 ref|NP_531453.1|3,4-dihydroxy-2- CK LL butanone-4-phoshate synthase/GTP cyclohydrolase II [Agrobacterium tumefaciens str. C58] 375 77195 0 94 gb|AAM71555.1|mannose-6- PP SS PEG LL phosphate isomerase/mannose-1- phosphate guanylyl transferase [Chlorobium tepidum TLS] 376 77124 1.00E−180 100 ref|NP_418404.1|biotin--protein PP SS PEG ligase [Escherichia coli K12] 377 77261 0 100 ref|NP_416982.1|hydrogenase 4, PEG subunit [Escherichia coli K12] 378 77273 0 93 dbj|BAB06534.1|glycine PP PEG dehydrogenase subunit 1 [Bacillus halodurans C-125] 379 77203 1.00E−176 60 dbj|BAD33942.1|putative serine PEG CK carboxypeptidase precursor [Oryza sativa (japonica cultivar-group)] 380 77275 4.00E−37 55 gb|ABA91490.1|expressed protein SS HS PEG [Oryza sativa (japonica cultivar- group)] 381 77204 2.00E−27 44 ref|XP_469963.1|putative protease CK CS inhibitor [Oryza sativa (japonica cultivar-group)] 382 77266 7.00E−25 73 gb|AAT93978.1|unknown protein CS PP PEG [Oryza sativa (japonica cultivar- group)] 383 77220 1.00E−169 78 dbj|BAD52854.1|putative non- LL phototropic hypocotyl 3 [Oryza sativa (japonica cultivar-group)] 384 77268 4.00E−71 85 gb|ABF95596.1|ETC complex I PP SS subunit conserved region family protein, expressed [Oryza sativa (japonica cultivar-group)] 385 77209 9.00E−86 85 ref|XP_479456.1|putative 60S PP LL ribosome subunit biogenesis protein [Oryza sativa (japonica cultivar- group)] 386 77269 2.00E−56 70 dbj|BAD32031.1|unknown protein SS [Oryza sativa (japonica cultivar- group)] 387 77451 0 71 ref|XP_475231.1|putative PP SS microtubule-associated protein [Oryza sativa (japonica cultivar-group)] 388 77452 1.00E−106 69 dbj|BAD69045.1|unknown protein PP [Oryza sativa (japonica cultivar- group)] 389 77430 1.00E−160 83 dbj|BAD33328.1|putative protein CS PP SS serine/threonine kinase [Oryza sativa (japonica cultivar-group)] 390 77432 0 86 ref|NP_849565.1|carbohydrate PP HS PEG transporter/nucleoside transporter/ sugar porter [Arabidopsis thaliana] gb|AAM19835.1| AT4g35300/F23E12_140 [Arabidopsis thaliana] 391 77433 0 100 gb|AAK59487.1|putative cleavage CK PP and polyadenylation specificity factor [Arabidopsis thaliana] 392 77444 0 96 ref|NP_199947.1|unknown protein CS PP [Arabidopsis thaliana] 393 77409 0 91 ref|NP_001032163.1|unknown protein PP [Arabidopsis thaliana] 394 12313 0 94 ref|NP_189150.1|QUA1 SP (QUASIMODO1); transferase, transferring glycosyl groups/ transferase, transferring hexosyl groups [Arabidopsis thaliana]

Trait Improvement Screens

DS—Improvement of drought tolerance identified by a soil drought stress tolerance screen: Drought or water deficit conditions impose mainly osmotic stress on plants. Plants are particularly vulnerable to drought during the flowering stage. The drought condition in the screening process disclosed in Example 1B started from the flowering time and was sustained to the end of harvesting. The present invention provides recombinant DNA that can improve the plant survival rate under such sustained drought condition. Exemplary recombinant DNA for conferring such drought tolerance are identified as such in Table 3. Such recombinant DNA may find particular use in generating transgenic plants that are tolerant to the drought condition imposed during flowering time and in other stages of the plant life cycle. As demonstrated from the model plant screen, in some embodiments of transgenic plants with trait-improving recombinant DNA grown under such sustained drought condition can also have increased total seed weight per plant in addition to the increased survival rate within a transgenic population, providing a higher yield potential as compared to control plants.

PEG-Improvement of drought tolerance identified by PEG induced osmotic stress tolerance screen: Various drought levels can be artificially induced by using various concentrations of polyethylene glycol (PEG) to produce different osmotic potentials (Pilon-Smits e.g., (1995) Plant Physiol. 107:125-130). Several physiological characteristics have been reported as being reliable indications for selection of plants possessing drought tolerance. These characteristics include the rate of seed germination and seedling growth. The traits can be assayed relatively easily by measuring the growth rate of seedling in PEG solution. Thus, a PEG-induced osmotic stress tolerance screen is a useful surrogate for drought tolerance screen. As demonstrated from the model plant screen, embodiments of transgenic plants with trait-improving recombinant DNA identified in the PEG-induced osmotic stress tolerance screen can survive better drought conditions providing a higher yield potential as compared to control plants.

SS-Improvement of drought tolerance identified by high salinity stress tolerance screen: Three different factors are responsible for salt damages: (1) osmotic effects, (2) disturbances in the mineralization process, and (3) toxic effects caused by the salt ions, e.g., inactivation of enzymes. While the first factor of salt stress results in the wilting of the plants that is similar to drought effect, the ionic aspect of salt stress is clearly distinct from drought. The present invention provides genes that help plants maintain biomass, root growth, and/or plant development in high salinity conditions, which are identified as such in Table 3. Since osmotic effect is one of the major components of salt stress, which is common to the drought stress, trait-improving recombinant DNA identified in a high salinity stress tolerance screen can also provide transgenic crops with improved drought tolerance. As demonstrated from the model plant screen, embodiments of transgenic plants with trait-improving recombinant DNA identified in a high salinity stress tolerance screen can survive better drought conditions and/or high salinity conditions providing a higher yield potential as compared to control plants.

HS-Improvement of drought tolerance identified by heat stress tolerance screen: Heat and drought stress often occur simultaneously, limiting plant growth. Heat stress can cause the reduction in photosynthesis rate, inhibition of leaf growth and osmotic potential in plants. Thus, genes identified by the present invention as heat stress tolerance conferring genes may also impart improved drought tolerance to plants. As demonstrated from the model plant screen, embodiments of transgenic plants with trait-improving recombinant DNA identified in a heat stress tolerance screen can survive better heat stress conditions and/or drought conditions providing a higher yield potential as compared to control plants.

CK and CS-Improvement of tolerance to cold stress: Low temperature may immediately result in mechanical constraints, changes in activities of macromolecules, and reduced osmotic potential. In the present invention, two screening conditions, i.e., cold shock tolerance screen (CK) and cold germination tolerance screen (CS), were set up to look for transgenic plants that display visual growth advantage at lower temperature. In cold germination tolerance screen, the transgenic Arabidopsis plants were exposed to a constant temperature of 8° C. from planting until day 28 post plating. The trait-improving recombinant DNA identified by such screen are particular useful for the production of transgenic plant that can germinate more robustly in a cold temperature as compared to the wild type plants. In cold shock tolerance screen, the transgenic plants were first grown under the normal growth temperature of 22° C. until day 8 post plating, and subsequently were placed under 8° C. until day 28 post plating. As demonstrated from the model plant screen, embodiments of transgenic plants with trait-improving recombinant DNA identified in a cold shock stress tolerance screen and/or a cold germination stress tolerance screen can survive better cold conditions providing a higher yield potential as compared to control plants.

Improvement of tolerance to multiple stresses: Different kinds of stresses often lead to identical or similar reaction in the plants. Genes that are activated or inactivated as a reaction to stress can either act directly in a way the genetic product reduces a specific stress, or they can act indirectly by activating other specific stress genes. By manipulating the activity of such regulatory genes, i.e., multiple stress tolerance genes, the plant can be enabled to react to different kinds of stresses. For examples, PEP SEQ ID NO: 231 can be used to improve both salt stress tolerance and cold stress tolerance in plants. Of particular interest, plants transformed with PEP SEQ ID NO: 233 can resist heat stress, salt stress and cold stress. In addition to these multiple stress tolerance genes, the stress tolerance conferring genes provided by the present invention may be used in combinations to generate transgenic plants that can resist multiple stress conditions.

PP-Improvement of early plant growth and development: It has been known in the art that to minimize the impact of disease on crop profitability, it is important to start the season with healthy and vigorous plants. This means avoiding seed and seedling diseases, leading to increased nutrient uptake and increased yield potential. Traditionally early planting and applying fertilizer are the methods used for promoting early seedling vigor. In early development stage, plant embryos establish only the basic root-shoot axis, a cotyledon storage organ(s), and stem cell populations, called the root and shoot apical meristems, that continuously generate new organs throughout post-embryonic development. “Early growth and development” used herein encompasses the stages of seed imbibition through the early vegetative phase. The present invention provides genes that are useful to produce transgenic plants that have advantages in one or more processes including, but not limited to, germination, seedling vigor, root growth and root morphology under non-stressed conditions. The transgenic plants starting from a more robust seedling are less susceptible to the fungal and bacterial pathogens that attach germinating seeds and seedling. Furthermore, seedlings with advantage in root growth are more resistant to drought stress due to extensive and deeper root architecture. Therefore, it can be recognized by those skilled in the art that genes conferring the growth advantage in early stages to plants may also be used to generate transgenic plants that are more resistant to various stress conditions due to improved early plant development. The present invention provides such exemplary recombinant DNA that confer both the stress tolerance and growth advantages to plants, identified as such in Table 3, e.g., PEP SEQ ID NO: 268 which can improve the plant early growth and development, and impart heat and cold tolerance to plants. As demonstrated from the model plant screen, embodiments of transgenic plants with trait-improving recombinant DNA identified in the early plant development screen can grow better under non-stress conditions and/or stress conditions providing a higher yield potential as compared to control plants.

SP-Improvement of late plant growth and development: “Late growth and development” used herein encompasses the stages of leaf development, flower production, and seed maturity. In certain embodiments, transgenic plants produced using genes that confer growth advantages to plants provided by the present invention, identified as such in Table 3, exhibit at least one phenotypic characteristics including, but not limited to, increased rosette radius, increased rosette dry weight, seed dry weight, silique dry weight, and silique length. On one hand, the rosette radius and rosette dry weight are used as the indexes of photosynthesis capacity, and thereby plant source strength and yield potential of a plant. On the other hand, the seed dry weight, silique dry weight and silique length are used as the indexes for plant sink strength, which are considered as the direct determinants of yield. As demonstrated from the model plant screen, embodiments of transgenic plants with trait-improving recombinant DNA identified in the late development screen can grow better and/or have improved development during leaf development and seed maturation providing a higher yield potential as compared to control plants.

LL-Improvement of tolerance to shade stress identified in a low light screen: The effects of light on plant development are especially prominent at the seedling stage. Under normal light conditions with unobstructed direct light, a plant seeding develops according to a characteristic photomorphogenic pattern, in which plants have open and expanded cotyledons and short hypocotyls. Then the plant's energy is devoted to cotyledon and leaf development while longitudinal extension growth is minimized. Under low light condition where light quality and intensity are reduced by shading, obstruction or high population density, a seedling displays a shade-avoidance pattern, in which the seedling displays a reduced cotyledon expansion, and hypocotyls extension is greatly increased. As the result, a plant under low light condition increases significantly its stem length at the expanse of leaf, seed or fruit and storage organ development, thereby adversely affecting of yield. The present invention provides recombinant DNA that enable plants to have an attenuated shade avoidance response so that the source of plant can be contributed to reproductive growth efficiently, resulting higher yield as compared to the wild type plants. As demonstrated from the model plant screen, embodiments of transgenic plants with trait-improving recombinant DNA identified in a shade stress tolerance screen can have attenuated shade response under shade conditions providing a higher yield potential as compared to control plants. The transgenic plants generated by the present invention may be suitable for a higher density planting, thereby resulting increased yield per unit area.

LN-Improvement of Tolerance to Low Nitrogen Availability Stress

Nitrogen is a key factor in plant growth and crop yield. The metabolism, growth and development of plants are profoundly affected by their nitrogen supply. Restricted nitrogen supply alters shoot to root ratio, root development, activity of enzymes of primary metabolism and the rate of senescence (death) of older leaves. All field crops have a fundamental dependence on inorganic nitrogenous fertilizer. Since fertilizer is rapidly depleted from most soil types, it must be supplied to growing crops two or three times during the growing season. Enhanced nitrogen use efficiency by plants should enable crops cultivated under low nitrogen availability stress condition resulted from low fertilizer input or poor soil quality.

According to the present invention, transgenic plants generated using the recombinant nucleotides, which confer enhanced nitrogen use efficiency, identified as such in Table 3, exhibit one or more desirable traits including, but not limited to, increased seedling weight, greener leaves, increased number of rosette leaves, increased or decreased root length. One skilled in the art may recognize that the transgenic plants provided by the present invention with enhanced nitrogen use efficiency may also have altered amino acid or protein compositions, increased yield and/or better seed quality. The transgenic plants of the present invention may be productively cultivated under low nitrogen growth conditions, i.e., nitrogen-poor soils and low nitrogen fertilizer inputs, which would cause the growth of wild type plants to cease or to be so diminished as to make the wild type plants practically useless. The transgenic plants also may be advantageously used to achieve earlier maturing, faster growing, and/or higher yielding crops and/or produce more nutritious foods and animal feedstocks when cultivated using nitrogen non-limiting growth conditions.

Stacked Traits: The present invention also encompasses transgenic plants with stacked engineered traits, e.g., a crop having an improved phenotype resulting from expression of a trait-improving recombinant DNA, in combination with herbicide and/or pest resistance traits. For example, genes of the current invention can be stacked with other traits of agronomic interest, such as a trait providing herbicide resistance, for example a RoundUp Ready® trait, or insect resistance, such as using a gene from Bacillus thuringensis to provide resistance against lepidopteran, coliopteran, homopteran, hemiopteran, and other insects. Herbicides for which resistance is useful in a plant include glyphosate herbicides, phosphinothricin herbicides, oxynil herbicides, imidazolinone herbicides, dinitroaniline herbicides, pyridine herbicides, sulfonylurea herbicides, bialaphos herbicides, sulfonamide herbicides and gluphosinate herbicides. To illustrate that the production of transgenic plants with herbicide resistance is a capability of those of ordinary skill in the art, reference is made to U.S. patent application publications 2003/0106096A1 and 2002/0112260A1 and U.S. Pat. Nos. 5,034,322; 5,776,760, 6,107,549 and 6,376,754, all of which are incorporated herein by reference. To illustrate that the production of transgenic plants with pest resistance is a capability of those of ordinary skill in the art reference is made to U.S. Pat. Nos. 5,250,515 and 5,880,275 which disclose plants expressing an endotoxin of Bacillus thuringiensis bacteria, to U.S. Pat. No. 6,506,599 which discloses control of invertebrates which feed on transgenic plants which express dsRNA for suppressing a target gene in the invertebrate, to U.S. Pat. No. 5,986,175 which discloses the control of viral pests by transgenic plants which express viral replicase, and to U.S. Patent Application Publication 2003/0150017 A1 which discloses control of pests by a transgenic plant which express a dsRNA targeted to suppressing a gene in the pest, all of which are incorporated herein by reference.

Once one recombinant DNA has been identified as conferring an improved trait of interest in transgenic Arabidopsis plants, several methods are available for using the sequence of that recombinant DNA and knowledge about the protein it encodes to identify homologs of that sequence from the same plant or different plant species or other organisms, e.g., bacteria and yeast. Thus, in one aspect, the invention provides methods for identifying a homologous gene with a DNA sequence homologous to any of SEQ ID NO: 1 through SEQ ID NO: 197, or a homologous protein with an amino acid sequence homologous to any of SEQ ID NO: 198 through SEQ ID NO: 394. In another aspect, the present invention provides the protein sequences of identified homologs for a sequence listed as SEQ ID NO: 395 through SEQ ID NO: 19938. In yet another aspect, the present invention also includes linking or associating one or more desired traits, or gene function with a homolog sequence provided herein.

The trait-improving recombinant DNA and methods of using such trait-improving recombinant DNA for generating transgenic plants with improved traits provided by the present invention are not limited to any particular plant species. Indeed, the plants according to the present invention may be of any plant species, i.e., may be monocotyledonous or dicotyledonous. Preferably, they will be agricultural useful plants, i.e., plants cultivated by man for purposes of food production or technical, particularly industrial applications. Of particular interest in the present invention are corn and soybean plants. The recombinant DNA constructs optimized for soybean transformation and recombinant DNA constructs optimized for corn transformation are provided by the present invention. Other plants of interest in the present invention for production of transgenic plants having improved traits include, without limitation, cotton, canola, wheat, sunflower, sorghum, alfalfa, barley, millet, rice, tobacco, fruit and vegetable crops, and turfgrass.

In certain embodiments, the present invention contemplates to use an orthologous gene in generating the transgenic plants with similarly improved traits as the transgenic Arabidopsis counterpart. Improved physiological properties in transgenic plants of the present invention may be confirmed in responses to stress conditions, for example in assays using imposed stress conditions to detect improved responses to drought stress, nitrogen deficiency, cold growing conditions, or alternatively, under naturally present stress conditions, for example under field conditions. Biomass measures may be made on greenhouse or field grown plants and may include such measurements as plant height, stem diameter, root and shoot dry weights, and, for corn plants, ear length and diameter.

Trait data on morphological changes may be collected by visual observation during the process of plant regeneration as well as in regenerated plants transferred to soil. Such trait data includes characteristics such as normal plants, bushy plants, taller plants, thicker stalks, narrow leaves, striped leaves, knotted phenotype, chlorosis, albino, anthocyanin production, or altered tassels, ears or roots. Other enhanced traits may be identified by measurements taken under field conditions, such as days to pollen shed, days to silking, leaf extension rate, chlorophyll content, leaf temperature, stand, seedling vigor, internode length, plant height, leaf number, leaf area, tillering, brace roots, stay green, stalk lodging, root lodging, plant health, barreness/prolificacy, green snap, and pest resistance. In addition, trait characteristics of harvested grain may be confirmed, including number of kernels per row on the ear, number of rows of kernels on the ear, kernel abortion, kernel weight, kernel size, kernel density and physical grain quality.

To confirm hybrid yield in transgenic corn plants expressing genes of the present invention, it may be desirable to test hybrids over multiple years at multiple locations in a geographical location where maize is conventionally grown, e.g., in Iowa, Illinois or other locations in the midwestern United States, under “normal” field conditions as well as under stress conditions, e.g., under drought or population density stress.

Transgenic plants can be used to provide plant parts according to the invention for regeneration or tissue culture of cells or tissues containing the constructs described herein. Plant parts for these purposes can include leaves, stems, roots, flowers, tissues, epicotyl, meristems, hypocotyls, cotyledons, pollen, ovaries, cells and protoplasts, or any other portion of the plant which can be used to regenerate additional transgenic plants, cells, protoplasts or tissue culture. Seeds of transgenic plants are provided by this invention can be used to propagate more plants containing the trait-improving recombinant DNA constructs of this invention. These descendants are intended to be included in the scope of this invention if they contain a trait-improving recombinant DNA construct of this invention, whether or not these plants are selfed or crossed with different varieties of plants.

The various aspects of the invention are illustrated by means of the following examples which are in no way intended to limit the full breath and scope of claims.

EXAMPLES Example 1 Identification of Recombinant DNA that Confers Improved Trait(s) to Plants A. Plant Expression Constructs for Arabidopsis Transformation

Each gene of interest was amplified from a genomic or cDNA library using primers specific to sequences upstream and downstream of the coding region. Transformation vectors were prepared to constitutively transcribe DNA in either sense orientation (for enhanced protein expression) or anti-sense orientation (for endogenous gene suppression) under the control of an enhanced Cauliflower Mosaic Virus 35S promoter (U.S. Pat. No. 5,359,142) directly or indirectly (Moore, e.g., PNAS 95:376-381, 1998; Guyer, e.g., Genetics 149: 633-639, 1998; International patent application NO. PCT/EP98/07577). The transformation vectors also contain a bar gene as a selectable marker for resistance to glufosinate herbicide. The transformation of Arabidopsis plants was carried out using the vacuum infiltration method known in the art (Bethtold, e.g., Methods Mol. Biol. 82:259-66, 1998). Seeds harvested from the plants, named as T1 seeds, were subsequently grown in a glufosinate-containing selective medium to select for plants which were actually transformed and which produced T2 transgenic seed.

B. Soil Drought Tolerance Screen

This example describes a soil drought tolerance screen to identify Arabidopsis plants transformed with recombinant DNA that wilt less rapidly and/or produce higher seed yield when grown in soil under drought conditions

T2 seeds were sown in flats filled with Metro/Mix® 200 (The Scotts® Company, USA). Humidity domes were added to each flat and flats were assigned locations and placed in climate-controlled growth chambers. Plants were grown under a temperature regime of 22° C. at day and 20° C. at night, with a photoperiod of 16 hours and average light intensity of 170 μmol/m²/s. After the first true leaves appeared, humidity domes were removed. The plants were sprayed with glufosinate herbicide and put back in the growth chamber for 3 additional days. Flats were watered for 1 hour the week following the herbicide treatment. Watering was continued every seven days until the flower bud primordia became apparent, at which time plants were watered for the last time.

To identify drought tolerant plants, plants were evaluated for wilting response and seed yield. Beginning ten days after the last watering, plants were examined daily until 4 plants/line had wilted. In the next six days, plants were monitored for wilting response. Five drought scores were assigned according to the visual inspection of the phenotypes: 1 for healthy, 2 for dark green, 3 for wilting, 4 severe wilting, and 5 for dead. A score of 3 or higher was considered as wilted.

At the end of this assay, seed yield measured as seed weight per plant under the drought condition was characterized for the transgenic plants and their controls and analyzed as a quantitative response according to example 1M.

Two approaches were used for statistical analysis on the wilting response. First, the risk score was analyzed for wilting phenotype and treated as a qualitative response according to the example 1L. Alternatively, the survival analysis was carried out in which the proportions of wilted and non-wilted transgenic and control plants were compared over each of the six days under scoring and an overall log rank test was performed to compare the two survival curves using S-PLUS statistical software (S-PLUS 6, Guide to statistics, Insightful, Seattle, Wash., USA). A list of recombinant DNA constructs which improve drought tolerance in transgenic plants is illustrated in Table 4.

TABLE 4 PEP Drought Time to wilting SEQ score Seed yield Risk ID Construct Nomination Delta P- Delta P- score P- NO ID ID Orientation mean value mean value mean value 198 11029 CGPG106 SENSE 0.109 0.381 0.072 0.717 0.119 1.000 292 73136 CGPG5764 SENSE 0.030 0.090 0.136 0.067 0.104 1.000 284 73165 CGPG5661 SENSE −0.031 0.504 0.526 0.018 −0.075 1.000 260 73628 CGPG5025 SENSE 0.342 0.038 −0.474 0.022 0.208 1.000 291 73981 CGPG5757 SENSE 0.517 0.026 −0.096 0.000 0.573 1.000 207 74065 CGPG1828 ANTI- −0.078 0.164 0.646 0.027 0.048 1.000 SENSE 263 74217 CGPG5144 SENSE −0.026 0.414 0.418 0.032 0.071 1.000 261 76105 CGPG5041 SENSE 0.288 0.029 −1.780 0.027 0.173 1.000 328 76576 CGPG7281 SENSE 0.241 0.032 −0.555 0.442 0.212 1.000 327 77536 CGPG7272 SENSE 0.089 0.194 1.059 0.001 0.042 1.000 226 19767 CGPG3918 SENSE 0.117 0.038 0.164 0.296 / / 237 19947 CGPG4069 SENSE −0.009 0.852 −0.104 0.504 / / 242 70994 CGPG4122 SENSE 0.040 0.021 −0.131 0.447 / / 363 77103 CGPG9134 SENSE 0.145 0.023 −0.499 0.096 / /

If p<0.05 and delta or risk score mean >0, the transgenic plants showed statistically significant trait improvement as compared to the reference (p value, of the delta of a quantitative response or of the risk score of a qualitative response, is the probability that the observed difference between the transgenic plants and the reference occur by chance) If p<0.2 and delta or risk score mean >0, the transgenic plants showed a trend of trait improvement as compared to the reference.

Transgenic plants comprising recombinant DNA expressing a protein as set forth in SEQ ID NO: 226, 237, 242, or 363 showed improved drought tolerance evidenced by the second criteria as illustrated in Example 1L and 1M.

C. Heat Stress Tolerance Screen

Under high temperatures, Arabidopsis seedlings become chlorotic and root growth is inhibited. This example sets forth the heat stress tolerance screen to identify Arabidopsis plants transformed with the gene of interest that are more resistant to heat stress based on primarily their seedling weight and root growth under high temperature.

T2 seeds were plated on ½ X MS salts, 1% phytagel, with 10 μg/ml BASTA (7 per plate with 2 control seeds; 9 seeds total per plate). Plates were placed at 4° C. for 3 days to stratify seeds. Plates were then incubated at room temperature for 3 hours and then held vertically for 11 additional days at temperature of 34° C. at day and 20° C. at night. Photoperiod was 16 h. Average light intensity was ˜140 μmol/m²/s. After 14 days of growth, plants were scored for glufosinate resistance, root length, final growth stage, visual color, and seedling fresh weight. A photograph of the whole plate was taken on day 14.

The seedling weight and root length were analyzed as quantitative responses according to example 1M. The final grow stage at day 14 was scored as success if 50% of the plants had reached 3 rosette leaves and size of leaves are greater than 1 mm (Boyes, e.g., (2001) The Plant Cell 13, 1499-1510). The growth stage data was analyzed as a qualitative response according to example 1L. A list of recombinant DNA constructs that improve heat tolerance in transgenic plants illustrated in Table 5.

TABLE 5 Growth stage Seedling Root length at day 14 weight PEP at day 14 Risk at day 14 SEQ Construct Nomination Delta score Delta ID ID ID Orientation mean P-value mean P-value mean P-value 239 70985 CGPG4088 SENSE 0.480 0.036 1.176 0.196 1.333 0.018 233 70987 CGPG4048 SENSE 0.228 0.021 −0.036 0.670 1.214 0.002 268 72001 CGPG5221 SENSE 0.273 0.004 0.210 0.330 1.393 0.009 274 72753 CGPG5540 SENSE 0.434 0.006 0.414 0.258 1.100 0.006 276 73954 CGPG5577 SENSE 0.406 0.002 0.455 0.282 1.274 0.000 325 76178 CGPG7225 SENSE 0.714 0.001 1.093 0.145 1.537 0.000 361 77150 CGPG9130 SENSE 0.219 0.032 0.178 0.237 1.159 0.002 367 77164 CGPG9147 SENSE 0.317 0.026 0.651 0.099 1.152 0.002 390 77432 CGPG9335 SENSE 0.581 0.001 0.457 0.124 1.551 0.000 351 77587 CGPG8107 SENSE 0.488 0.006 0.628 0.072 1.328 0.001 348 77928 CGPG8082 SENSE 0.462 0.032 0.191 0.301 1.498 0.004 212 16610 CGPG2499 SENSE 0.055 0.640 0.109 0.609 0.983 0.020 220 18256 CGPG3363 SENSE 0.070 0.138 0.071 0.541 1.021 0.004 222 19193 CGPG3375 SENSE 0.255 0.094 0.267 0.368 1.023 0.003 252 70684 CGPG4588 SENSE 0.010 0.781 0.003 0.979 1.002 0.045 230 70948 CGPG3990 SENSE 0.282 0.095 0.351 0.335 1.045 0.023 243 70956 CGPG4140 SENSE 0.155 0.313 0.091 0.320 1.129 0.018 235 70962 CGPG4057 SENSE 0.469 0.085 0.267 0.508 1.410 0.007 241 70963 CGPG4121 SENSE −0.002 0.979 −0.131 0.311 0.976 0.005 242 70994 CGPG4122 SENSE 0.140 0.574 1.204 0.096 1.207 0.030 244 70995 CGPG4154 SENSE 0.374 0.112 1.640 0.171 1.328 0.013 208 72783 CGPG2206 SENSE 0.387 0.057 0.053 0.509 1.013 0.019 255 72807 CGPG4912 SENSE 0.357 0.071 0.443 0.020 1.099 0.009 259 72825 CGPG5001 SENSE 0.171 0.349 0.213 0.234 1.118 0.012 287 73120 CGPG5704 SENSE 0.096 0.490 0.193 0.364 1.080 0.033 280 73127 CGPG5640 SENSE −0.220 0.077 −0.194 0.030 0.643 0.014 293 73172 CGPG5783 SENSE 0.283 0.197 0.887 0.162 1.154 0.007 310 73411 CGPG6440 SENSE 0.198 0.389 0.631 0.345 1.320 0.003 260 73628 CGPG5025 SENSE 0.406 0.149 1.475 0.365 1.169 0.016 316 74566 CGPG6796 SENSE 0.627 0.092 0.777 0.466 1.714 0.014 338 75654 CGPG7804 SENSE 0.292 0.237 0.804 0.315 0.940 0.017 340 75657 CGPG7828 SENSE 0.390 0.067 1.222 0.307 1.521 0.011 213 76602 CGPG2653 SENSE 0.036 0.748 0.092 0.670 0.752 0.025 372 77180 CGPG9180 SENSE 0.068 0.555 −0.045 0.177 0.871 0.022 380 77275 CGPG9236 SENSE 0.282 0.125 0.971 0.285 1.027 0.013 297 77312 CGPG5927 SENSE 0.201 0.344 0.710 0.372 1.039 0.032 320 77518 CGPG6953 SENSE 0.238 0.344 0.069 0.655 1.188 0.015 342 77549 CGPG7933 SENSE 0.184 0.180 −0.012 / 0.978 0.007 347 77580 CGPG8062 SENSE 0.157 0.143 −0.074 / 0.891 0.049 317 77610 CGPG6805 SENSE 0.023 0.937 −0.063 / 1.207 0.029 318 77618 CGPG6810 SENSE 0.002 0.942 −0.074 / 1.015 0.006 353 77619 CGPG8152 SENSE 0.213 0.316 −0.076 0.457 0.956 0.019 355 77629 CGPG8377 SENSE 0.095 0.124 −0.063 / 1.133 0.005 334 77816 CGPG7529 SENSE 0.033 0.705 0.856 0.385 0.611 0.038

If p<0.05 and delta or risk score mean >0, the transgenic plants showed statistically significant trait improvement as compared to the reference. If p<0.2 and delta or risk score mean >0, the transgenic plants showed a trend of trait improvement as compared to the reference.

Transgenic plants comprising recombinant DNA expressing a protein as set forth in SEQ ID NO: 237, 307, 313, 327, 330, 349, 366, or 387 showed improved heat stress tolerance evidenced by the second criteria as illustrated in Example 1L and 1M.

D. Salt Stress Tolerance Screen

This example sets forth the high salinity stress screen to identify Arabidopsis plants transformed with the gene of interest that are tolerant to high levels of salt based on their rate of development, root growth and chlorophyll accumulation under high salt conditions.

T2 seeds were plated on glufosinate selection plates containing 90 mM NaCl and grown under standard light and temperature conditions. All seedlings used in the experiment were grown at a temperature of 22° C. at day and 20° C. at night, a 16-hour photoperiod, an average light intensity of approximately 120 umol/m². On day 11, plants were measured for primary root length. After 3 more days of growth (day 14), plants were scored for transgenic status, primary root length, growth stage, visual color, and the seedlings were pooled for fresh weight measurement. A photograph of the whole plate was also taken on day 14.

The seedling weight and root length were analyzed as quantitative responses according to example 1M. The final growth stage at day 14 was scored as success if 50% of the plants reached 3 rosette leaves and size of leaves are greater than 1 mm (Boyes, D. C., et al., (2001), The Plant Cell 13, 1499/1510). The growth stage data was analyzed as a qualitative response according to example 1L. A list of recombinant DNA constructs that improve high salinity tolerance in transgenic plants illustrated in Table 6.

TABLE 6 Root Root Growth Seedling length length stage weight PEP at day 11 at day 14 at day 14 at day 14 SEQ Construct Delta P- Delta P- Delta P- Delta P- ID ID Orientation mean value mean value mean value mean value 220 18256 SENSE 0.192 0.232 0.236 0.080 1.291 0.151 0.598 0.039 214 18456 SENSE 0.213 0.089 0.209 0.197 2.062 0.197 0.476 0.046 232 19973 SENSE 0.289 0.012 0.299 0.002 0.976 0.054 1.002 0.013 231 70201 SENSE 0.363 0.030 0.403 0.052 2.083 0.229 1.057 0.014 243 70956 SENSE 0.006 0.923 0.128 0.016 −0.045 NA −0.364 0.371 238 70969 SENSE 0.435 0.091 0.471 0.027 0.653 0.473 0.994 0.058 240 70971 SENSE 0.337 0.063 0.257 0.041 1.418 0.226 0.593 0.030 233 70987 SENSE 0.363 0.030 0.284 0.044 1.434 0.380 0.865 0.012 273 72788 SENSE 0.484 0.019 0.456 0.016 1.813 0.314 0.929 0.008 256 72811 SENSE 0.385 0.024 0.409 0.002 2.968 0.031 0.805 0.004 287 73120 SENSE 0.341 0.001 0.244 0.019 0.613 0.103 0.755 0.015 280 73127 SENSE 0.501 0.028 0.395 0.055 0.212 0.175 0.739 0.023 289 73134 SENSE 0.226 0.134 0.266 0.045 0.152 0.487 0.378 0.056 257 73235 SENSE 0.431 0.020 0.360 0.018 1.618 0.327 0.650 0.009 245 73306 SENSE 0.228 0.046 0.219 0.018 0.249 0.217 0.708 0.008 253 73330 SENSE 0.196 0.225 0.186 0.029 0.000 NA 0.543 0.045 316 74566 SENSE 0.331 0.001 0.076 0.422 0.597 0.031 0.716 0.003 312 74688 SENSE 0.480 0.076 0.415 0.031 0.000 NA 0.808 0.062 376 77124 SENSE 0.340 0.034 0.322 0.015 2.306 0.137 0.411 0.168 364 77139 SENSE 0.339 0.096 0.287 0.041 1.683 0.294 0.602 0.039 375 77195 SENSE 0.445 0.009 0.345 0.010 0.367 0.185 0.582 0.022 384 77268 SENSE 0.495 0.047 0.468 0.102 1.906 0.079 0.992 0.042 386 77269 SENSE 0.454 0.038 0.426 0.004 1.996 0.065 1.136 0.001 380 77275 SENSE 0.457 0.008 0.487 0.040 2.119 0.154 1.070 0.010 297 77312 SENSE 0.335 0.086 0.361 0.013 0.960 0.208 0.723 0.063 389 77430 SENSE 0.303 0.030 0.360 0.004 0.131 0.495 0.894 0.022 320 77518 SENSE 0.171 0.017 0.185 0.026 −0.134 0.443 0.339 0.109 345 77570 SENSE 0.326 0.091 0.241 0.134 1.396 0.090 0.862 0.026 351 77587 SENSE 0.178 0.083 0.096 0.665 0.995 0.426 0.632 0.036 353 77619 SENSE 0.389 0.058 0.398 0.011 1.913 0.257 0.857 0.040 355 77629 SENSE 0.404 0.043 0.389 0.019 2.523 0.083 0.826 0.040 336 77821 SENSE 0.393 0.063 0.444 0.033 0.196 0.228 0.619 0.015 356 77832 SENSE 0.134 0.238 0.304 0.011 0.914 0.156 0.413 0.050 352 77933 SENSE 0.112 0.044 0.165 0.009 0.749 0.482 0.517 0.171 347 77580 SENSE −0.019 0.814 0.087 0.018 −0.158 0.151 0.124 0.204

If p<0.05 and delta or risk score mean >0, the transgenic plants showed statistically significant trait improvement as compared to the reference. If p<0.2 and delta or risk score mean >0, the transgenic plants showed a trend of trait improvement as compared to the reference.

Transgenic plants comprising recombinant DNA expressing a protein as set forth in SEQ ID NO: 237, 242, 255, 307, 313, 327, 330, 349, 366, or 387 showed improved salt stress tolerance evidenced by the second criteria as illustrated in Example 1L.

E. Polyethylene Glycol (PEG) Induced Osmotic Stress Tolerance Screen

There are numerous factors, which can influence seed germination and subsequent seedling growth, one being the availability of water. Genes, which can directly affect the success rate of germination and early seedling growth, are potentially useful agronomic traits for improving the germination and growth of crop plants under drought stress. In this assay, PEG was used to induce osmotic stress on germinating transgenic lines of Arabidopsis thaliana seeds in order to screen for osmotically resistant seed lines.

T2 seeds were plated on BASTA selection plates containing 3% PEG and grown under standard light and temperature conditions. Seeds were plated on each plate containing 3% PEG, ½ X MS salts, 1% phytagel, and 10 μg/ml glufosinate. Plates were placed at 4° C. for 3 days to stratify seeds. On day 11, plants were measured for primary root length. After 3 more days of growth, i.e., at day 14, plants were scored for transgenic status, primary root length, growth stage, visual color, and the seedlings were pooled for fresh weight measurement. A photograph of the whole plate was taken on day 14.

Seedling weight and root length were analyzed as quantitative responses according to example 1M. The final growth stage at day 14 was scored as success or failure based on whether the plants reached 3 rosette leaves and size of leaves are greater than 1 mm. The growth stage data was analyzed as a qualitative response according to example 1L. A list of recombinant DNA constructs that improve osmotic stress tolerance in transgenic plants illustrated in Table 7.

TABLE 7 Root Root Growth Seedling length length stage weight PEP at day 11 at day 14 at day 14 at day 14 SEQ Construct Delta P- Delta P- Delta P- Delta P- ID ID Orientation mean value mean value mean value mean value 199 12223 SENSE / / / / / / 0.353 0.024 214 18456 SENSE 0.320 0.018 0.274 0.033 3.109 0.073 0.553 0.006 302 70812 SENSE 0.195 0.078 0.078 0.296 4 0.000 0.363 0.016 234 70950 SENSE 0.322 0.056 0.427 0.016 4 0.000 0.381 0.127 239 70985 SENSE 0.270 0.066 0.221 0.005 4 0.000 0.372 0.007 233 70987 SENSE 0.227 0.205 0.128 0.470 4 0.000 0.495 0.000 228 70992 SENSE 0.369 0.002 0.370 0.013 4 0.000 0.574 0.027 244 70995 SENSE 0.379 0.049 0.320 0.065 4 0.000 0.491 0.003 208 72783 SENSE 0.154 0.084 0.043 0.553 4 0.000 0.264 0.044 255 72807 SENSE 0.165 0.080 0.158 0.150 4 0.000 0.333 0.047 256 72811 SENSE 0.489 0.052 0.491 0.050 4 0.000 0.784 0.016 259 72825 SENSE 0.423 0.055 0.388 0.080 2.741 0.161 0.608 0.020 295 72946 SENSE 0.345 0.053 0.473 0.050 0.898 0.632 0.415 0.181 286 73026 SENSE 0.636 0.021 0.619 0.028 2.741 0.161 0.752 0.045 290 73123 SENSE 0.460 0.048 0.445 0.007 NA NA 0.609 0.069 278 73161 SENSE 0.510 0.024 0.506 0.006 2.624 0.246 0.626 0.073 284 73165 SENSE 0.304 0.101 0.285 0.044 2.499 0.270 0.643 0.077 293 73172 SENSE 0.197 0.056 0.137 0.204 2.374 0.293 0.660 0.081 262 73242 SENSE 0.305 0.029 0.332 0.022 2.249 0.317 0.677 0.085 265 73256 SENSE 0.325 0.084 0.182 0.178 2.124 0.341 0.694 0.089 308 73485 SENSE 0.133 0.070 0.039 0.583 1.999 0.365 0.711 0.093 291 73981 SENSE 0.239 0.147 0.238 0.281 1.874 0.389 0.728 0.096 316 74566 SENSE 0.451 0.004 0.248 0.118 1.749 0.412 0.745 0.100 312 74688 SENSE 0.190 0.191 0.102 0.520 1.624 0.436 0.762 0.104 330 74863 SENSE 0.174 0.164 0.216 0.109 1.499 0.460 0.779 0.108 366 77140 SENSE 0.644 0.005 0.572 0.015 1.374 0.484 0.796 0.112 361 77150 SENSE 0.313 0.047 0.359 0.044 1.249 0.508 0.813 0.116 365 77187 SENSE 0.273 0.084 0.032 0.763 1.124 0.532 0.830 0.120 375 77195 SENSE 0.319 0.021 0.256 0.041 NA NA 0.847 0.124 379 77203 SENSE 0.113 0.485 0.019 0.829 0.999 0.555 0.864 0.127 377 77261 SENSE 0.115 0.230 0.085 0.315 0.874 0.579 0.881 0.131 270 77308 SENSE 0.174 0.321 0.074 0.700 0.749 0.603 0.898 0.135 346 77338 SENSE 0.227 0.158 0.196 0.272 0.624 0.627 0.915 0.139 350 77357 SENSE 0.169 0.300 0.259 0.066 2.715 0.169 0.303 0.020 390 77432 SENSE 0.306 0.043 0.122 0.270 2.57 0.214 0.734 0.019 347 77580 SENSE 0.300 0.107 0.240 0.009 2.288 0.119 0.500 0.179 353 77619 SENSE 0.142 0.216 0.139 0.002 4 0.000 0.129 0.582 356 77832 SENSE 0.236 0.006 0.266 0.001 1.333 0.435 0.286 0.037 343 77917 SENSE 0.174 0.163 0.160 0.006 4 0.000 0.330 0.009

If p<0.05 and delta or risk score mean >0, the transgenic plants showed statistically significant trait improvement as compared to the reference.

If p<0.2 and delta or risk score mean >0, the transgenic plants showed a trend of trait improvement as compared to the reference.

Transgenic plants comprising recombinant DNA expressing a protein as set forth in SEQ ID NO: 209, 245, 260, 274, 282, 310, 321, 325, 336, 372, 376, 378, 380, or 382 showed improved osmotic stress tolerance evidenced by the second criteria as illustrated in Example 1L and 1M.

F. Cold Shock Tolerance Screen

This example set forth a screen to identify Arabidopsis plants transformed with the genes of interest that are more tolerant to cold stress subjected during day 8 to day 28 after seed planting. During these crucial early stages, seedling growth and leaf area increase were measured to assess tolerance when Arabidopsis seedlings were exposed to low temperatures. Using this screen, genetic alterations can be found that enable plants to germinate and grow better than wild type plants under sudden exposure to low temperatures.

Eleven seedlings from T2 seeds of each transgenic line plus one control line were plated together on a plate containing ½ X Gamborg Salts with 0.8 Phytagel™, 1% Phytagel, and 0.3% Sucrose. Plates were then oriented horizontally and stratified for three days at 4° C. At day three, plates were removed from stratification and exposed to standard conditions (16 hr photoperiod, 22° C. at day and 20° C. at night) until day 8. At day eight, plates were removed from standard conditions and exposed to cold shock conditions (24 hr photoperiod, 8° C. at both day and night) until the final day of the assay, i.e., day 28. Rosette areas were measured at day 8 and day 28, which were analyzed as quantitative responses according to example 1M. A list of recombinant nucleotides that improve cold shock stress tolerance in plants illustrated in Table 8.

TABLE 8 Rosette area Rosette area at day 28 Rosette area PEP at day 8 Risk difference SEQ Construct Nomination Delta P- score P- Delta P- ID ID ID Orientation mean value mean value mean value 199 12223 CGPG1133 SENSE 0.112 0.617 0.334 0.014 0.273 0.143 218 18231 CGPG3274 SENSE −0.131 0.269 1.144 0.002 1.179 0.003 215 18414 CGPG3002 SENSE 0.997 0.034 1.665 0.001 1.895 0.001 214 18456 CGPG2813 SENSE 0.099 0.757 0.799 0.001 0.684 0.001 233 70987 CGPG4048 SENSE 0.864 0.136 1.213 0.010 1.146 0.002 244 70995 CGPG4154 SENSE 1.088 0.008 1.262 0.023 1.407 0.025 208 72783 CGPG2206 SENSE 0.814 0.023 0.957 0.018 0.899 0.052 273 72788 CGPG5535 SENSE 0.262 0.058 1.090 0.016 1.212 0.021 256 72811 CGPG4926 SENSE 0.880 0.058 0.669 0.002 0.715 0.018 259 72825 CGPG5001 SENSE 0.032 0.887 0.338 0.071 0.357 0.027 281 73033 CGPG5646 SENSE 0.322 0.394 1.311 0.012 1.480 0.017 293 73172 CGPG5783 SENSE 0.395 0.209 1.065 0.030 1.099 0.074 253 73330 CGPG4765 SENSE 0.347 0.515 0.835 0.065 1.043 0.007 264 73735 CGPG5171 SENSE 0.624 0.076 0.655 0.001 0.718 0.005 315 74460 CGPG6747 SENSE 0.452 0.005 0.542 0.043 0.455 0.154 296 74746 CGPG5856 SENSE 0.304 0.346 0.978 0.001 1.061 0.007 299 75240 CGPG5957 SENSE 0.479 0.153 0.652 0.143 0.875 0.039 338 75654 CGPG7804 SENSE 0.531 0.140 1.712 0.010 1.979 0.015 339 75692 CGPG7823 SENSE 0.526 0.063 2.027 0.001 2.246 0.001 323 76161 CGPG7168 SENSE 0.768 0.000 1.810 0.004 2.146 0.003 217 76532 CGPG3235 SENSE 0.921 0.008 1.216 0.001 1.324 0.002 357 76802 CGPG8987 SENSE 0.468 0.097 1.578 0.000 1.872 0.000 358 76829 CGPG9013 SENSE −0.434 0.329 0.709 0.010 0.314 0.118 373 77121 CGPG9183 SENSE 0.081 0.385 0.757 0.023 0.698 0.014 366 77140 CGPG9145 SENSE −0.221 0.165 0.896 0.007 1.043 0.005 374 77157 CGPG9186 SENSE 0.190 0.566 1.110 0.016 1.176 0.015 369 77165 CGPG9155 SENSE 1.573 0.043 0.683 0.038 0.699 0.022 370 77166 CGPG9163 SENSE 0.777 0.011 1.432 0.016 1.570 0.025 381 77204 CGPG9238 SENSE 0.545 0.139 1.313 0.003 1.436 0.006 391 77433 CGPG9341 SENSE 1.054 0.103 0.895 0.027 0.659 0.084 345 77570 CGPG8015 SENSE 0.538 0.215 0.866 0.025 0.898 0.023 353 77619 CGPG8152 SENSE −0.589 0.322 0.503 0.068 0.558 0.049 354 77621 CGPG8166 SENSE 0.053 0.863 0.716 0.044 0.792 0.040 203 13411 CGPG1301 SENSE 0.183 0.427 0.577 0.033 0.573 0.031 209 17210 CGPG2217 SENSE −0.244 0.685 0.162 0.038 0.156 0.104 220 18256 CGPG3363 SENSE 0.272 0.400 0.639 0.008 0.719 0.020 289 73134 CGPG5721 SENSE 0.014 0.947 1.053 0.000 1.062 0.025 263 74217 CGPG5144 SENSE 0.168 0.390 0.261 0.043 0.206 0.075 379 77203 CGPG9230 SENSE 0.606 0.051 0.620 0.032 0.778 0.039

If p<0.05 and delta or risk score mean >0, the transgenic plants showed statistically significant trait improvement as compared to the reference (p value, of the delta of a quantitative response or of the risk score of a qualitative response, is the probability that the observed difference between the transgenic plants and the reference occur by chance)

If p<0.2 and delta or risk score mean >0, the transgenic plants showed a trend of trait improvement as compared to the reference.

Transgenic plants comprising recombinant DNA expressing a protein as set forth in SEQ ID NO: 255, 272, or 330 showed improved cold stress tolerance evidenced by the second criterial as illustrated in Example 1L.

G. Cold Germination Tolerance Screen

This example sets forth a screen to identify Arabidopsis plants transformed with the genes of interests are resistant to cold stress based on their rate of development, root growth and chlorophyll accumulation under low temperature conditions.

T2 seeds were plated and all seedlings used in the experiment were grown at 8° C. Seeds were first surface disinfested using chlorine gas and then seeded on assay plates containing an aqueous solution of ½ X Gamborg's B/5 Basal Salt Mixture (Sigma/Aldrich Corp., St. Louis, Mo., USA G/5788), 1% Phytagel™ (Sigma-Aldrich, P-8169), and 10 ug/ml glufosinate with the final pH adjusted to 5.8 using KOH. Test plates were held vertically for 28 days at a constant temperature of 8° C., a photoperiod of 16 hr, and average light intensity of approximately 100 umol/m²/s. At 28 days post plating, root length was measured, growth stage was observed, the visual color was assessed, and a whole plate photograph was taken.

The root length at day 28 was analyzed as a quantitative response according to example 1M. The growth stage at day 7 was analyzed as a qualitative response according to example 1L. A list of recombinant DNA constructs that improve cold stress tolerance in transgenic plants illustrated in Table 9.

TABLE 9 Root length Growth stage PEP at day 28 at day 28 SEQ Nomination Delta Delta ID Construct ID ID Orientation mean P-value mean P-value 209 17210 CGPG2217 SENSE 0.164 0.089 4.000 0.000 219 18232 CGPG3275 SENSE −0.081 0.613 4.000 0.000 226 19767 CGPG3918 SENSE 0.212 0.007 4.000 0.000 227 19774 CGPG3920 SENSE 0.140 0.011 4.000 0.000 237 19947 CGPG4069 SENSE / / 4.000 0.000 231 70201 CGPG3994 SENSE 0.311 0.010 4.000 0.000 252 70684 CGPG4588 SENSE 0.335 0.209 4.000 0.000 236 70915 CGPG4058 SENSE 0.231 0.258 4.000 0.000 243 70956 CGPG4140 SENSE 0.113 0.525 4.000 0.000 241 70963 CGPG4121 SENSE 0.210 0.252 4.000 0.000 239 70985 CGPG4088 SENSE 0.283 0.063 4.000 0.000 233 70987 CGPG4048 SENSE 0.392 0.202 4.000 0.000 228 70992 CGPG3962 SENSE 0.008 0.959 4.000 0.000 244 70995 CGPG4154 SENSE 0.426 0.002 4.000 0.000 268 72001 CGPG5221 SENSE 0.070 0.728 4.000 0.000 274 72753 CGPG5540 SENSE 0.189 0.010 2.889 0.121 273 72788 CGPG5535 SENSE 0.548 0.009 4.000 0.000 287 73120 CGPG5704 SENSE 0.401 0.146 4.000 0.000 290 73123 CGPG5728 SENSE 0.171 0.166 4.000 0.000 277 73137 CGPG5587 SENSE 0.382 0.019 4.000 0.000 283 73141 CGPG5659 SENSE 0.348 0.029 1.891 0.219 285 73155 CGPG5684 SENSE 0.194 0.105 4.000 0.000 278 73161 CGPG5594 SENSE 0.327 0.018 2.889 0.121 265 73256 CGPG5194 SENSE 0.176 0.473 4.000 0.000 266 73260 CGPG5200 SENSE 0.256 0.047 4.000 0.000 253 73330 CGPG4765 SENSE 0.229 0.016 4.000 0.000 310 73411 CGPG6440 SENSE 0.232 0.213 4.000 0.000 260 73628 CGPG5025 SENSE 0.304 0.040 1.330 0.467 204 73944 CGPG1458 SENSE 0.426 0.026 4.000 0.000 207 74065 CGPG1828 ANTI-SENSE 0.215 0.026 2.599 0.205 248 74536 CGPG442 SENSE −0.054 0.431 4.000 0.000 312 74688 CGPG6653 SENSE 0.645 0.006 4.000 0.000 340 75657 CGPG7828 SENSE 0.394 0.017 2.680 0.180 325 76178 CGPG7225 SENSE 0.334 0.107 4.000 0.000 321 76460 CGPG7121 SENSE 0.136 0.381 4.000 0.000 305 76527 CGPG6306 SENSE 0.238 0.001 0.335 0.421 217 76532 CGPG3235 SENSE 0.250 0.331 4.000 0.000 213 76602 CGPG2653 SENSE 0.283 0.387 4.000 0.000 306 77020 CGPG6318 SENSE 0.507 0.038 0.000 0.000 364 77139 CGPG9137 SENSE 0.445 0.005 0.000 0.000 372 77180 CGPG9180 SENSE 0.370 0.046 4.000 0.000 365 77187 CGPG9141 SENSE 0.456 0.036 4.000 0.000 381 77204 CGPG9238 SENSE 0.314 0.002 4.000 0.000 382 77266 CGPG9259 SENSE 0.017 0.948 4.000 0.000 389 77430 CGPG9322 SENSE 0.095 0.233 4.000 0.000 392 77444 CGPG9344 SENSE 0.171 0.292 4.000 0.000 319 77517 CGPG6952 SENSE 0.132 0.001 2.507 0.235 327 77536 CGPG7272 SENSE −0.084 0.675 4.000 0.000 351 77587 CGPG8107 SENSE 0.415 0.112 4.000 0.000 317 77610 CGPG6805 SENSE 0.267 0.180 4.000 0.000 356 77832 CGPG8976 SENSE 0.153 0.579 4.000 0.000

If p<0.05 and delta or risk score mean >0, the transgenic plants showed statistically significant trait improvement as compared to the reference. If p<0.2 and delta or risk score mean >0, the transgenic plants showed a trend of trait improvement as compared to the reference.

Transgenic plants comprising recombinant DNA expressing a protein as set forth in SEQ ID NO: 347 showed improved cold stress tolerance evidenced by the second criteria as illustrated in Example 1L and 1M.

H. Shade Tolerance Screen

Plants undergo a characteristic morphological response in shade that includes the elongation of the petiole, a change in the leaf angle, and a reduction in chlorophyll content. While these changes may confer a competitive advantage to individuals, in a monoculture the shade avoidance response is thought to reduce the overall biomass of the population. Thus, genetic alterations that prevent the shade avoidance response may be associated with higher yields. Genes that favor growth under low light conditions may also promote yield, as inadequate light levels frequently limit yield. This protocol describes a screen to look for Arabidopsis plants that show an attenuated shade avoidance response and/or grow better than control plants under low light intensity. Of particular interest, we were looking for plants that didn't extend their petiole length, had an increase in seedling weight relative to the reference and had leaves that were more close to parallel with the plate surface.

T2 seeds were plated on glufosinate selection plates with ½ MS medium. Seeds were sown on ½ X MS salts, 1% Phytagel, 10 ug/ml BASTA. Plants were grown on vertical plates at a temperature of 22° C. at day, 20° C. at night and under low light (approximately 30 uE/m²/s, far/red ratio (655/665/725/735) ˜0.35 using PLAQ lights with GAM color filter #680). Twenty-three days after seedlings were sown, measurements were recorded including seedling status, number of rosette leaves, status of flower bud, petiole leaf angle, petiole length, and pooled fresh weights. A digital image of the whole plate was taken on the measurement day. Seedling weight and petiole length were analyzed as quantitative responses according to example 1M. The number of rosette leaves, flowering bud formation and leaf angel were analyzed as qualitative responses according to example 1L.

A list of recombinant DNA constructs that improve shade tolerance in plants illustrated in Table 10.

TABLE 10 Leaf Seedling angle weight Petiole length PEP at day 23 at day 23 at day 23 SEQ Construct Nomination RS P- Delta P- Delta P- ID ID ID Orientation mean value mean value mean value 251 70755 CGPG4473 SENSE NA NA −0.751 0.017 −0.307 0.059 244 70995 CGPG4154 SENSE NA NA 0.251 0.070 0.008 0.944 275 72709 CGPG5568 SENSE NA NA −0.903 0.070 −0.608 0.019 210 72724 CGPG2292 SENSE NA NA −0.707 0.098 −0.638 0.020 272 72774 CGPG5518 SENSE NA NA −0.268 0.110 −0.195 0.087 258 72813 CGPG4977 SENSE NA NA −0.469 0.092 −0.186 0.054 294 73020 CGPG5791 SENSE NA NA −1.443 0.010 −1.265 0.010 288 73133 CGPG5714 SENSE NA NA −0.852 0.009 −0.896 0.012 260 73628 CGPG5025 SENSE NA NA 0.311 0.011 0.317 0.146 271 73766 CGPG5432 SENSE NA NA −0.988 0.048 −0.671 0.088 314 74435 CGPG6737 SENSE NA NA −0.735 0.042 −0.755 0.049 331 74858 CGPG7371 SENSE NA NA −0.667 0.010 −0.808 0.008 330 74863 CGPG7316 SENSE NA NA 0.176 0.060 0.168 0.515 332 74933 CGPG7457 SENSE NA NA −0.100 0.801 −0.533 0.094 340 75657 CGPG7828 SENSE NA NA 0.193 0.059 0.244 0.085 337 75685 CGPG7767 SENSE NA NA −0.917 0.075 −0.974 0.049 267 75822 CGPG5210 SENSE NA NA −0.267 0.194 −0.297 0.090 324 76171 CGPG7206 SENSE NA NA −0.350 0.065 −0.570 0.049 254 76202 CGPG4788 SENSE NA NA −1.153 0.009 −1.713 0.019 326 76467 CGPG7267 SENSE NA NA −0.484 0.177 −0.743 0.088 359 76961 CGPG9080 SENSE NA NA −0.425 0.098 −0.265 0.051 360 76973 CGPG9081 SENSE NA NA 0.333 0.052 0.248 0.003 363 77103 CGPG9134 SENSE NA NA 0.363 0.093 0.198 0.072 374 77157 CGPG9186 SENSE NA NA 0.287 0.044 0.107 0.086 369 77165 CGPG9155 SENSE NA NA −0.081 0.413 −0.572 0.012 368 77176 CGPG9148 SENSE NA NA −0.577 0.025 −0.651 0.085 365 77187 CGPG9141 SENSE NA NA 0.335 0.002 0.263 0.046 385 77209 CGPG9278 SENSE NA NA 0.671 0.027 0.451 0.011 383 77220 CGPG9271 SENSE NA NA 0.463 0.008 0.231 0.228 225 77334 CGPG3638 SENSE NA NA 0.531 0.067 0.336 0.031 349 77349 CGPG8083 SENSE NA NA 0.133 0.001 0.035 0.781 319 77517 CGPG6952 SENSE NA NA −0.843 0.142 −1.458 0.062 320 77518 CGPG6953 SENSE NA NA 0.354 0.007 0.380 0.010 307 77609 CGPG6326 SENSE NA NA 0.252 0.087 0.211 0.145

For “seeding weight” and “leaf angle”, if p<0.05 and delta or risk score mean >0, the transgenic plants showed statistically significant trait improvement as compared to the reference. If p<0.2 and delta or risk score mean >0, the transgenic plants showed a trend of trait improvement as compared to the reference with p<0.2.

For “petiole length”, if p<0.05 and delta <0, the transgenic plants showed statistically significant trait improvement as compared to the reference. If p<0.2 and delta <0, the transgenic plants showed a trend of trait improvement as compared to the reference.

Transgenic plants comprising recombinant DNA expressing a protein as set forth in SEQ ID NO: 246, 295, 303, 325, or 375 showed enhanced shade tolerance by the second criteria as illustrated in Example 1L and 1M.

I. Early Plant Growth and Development Screen

This example sets forth a plate based phenotypic analysis platform for the rapid detection of phenotypes that are evident during the first two weeks of growth. In this screen, we were looking for genes that confer advantages in the processes of germination, seedling vigor, root growth and root morphology under non-stressed growth conditions to plants. The transgenic plants with advantages in seedling growth and development were determined by the seedling weight and root length at day 14 after seed planting.

T2 seeds were plated on glufosinate selection plates and grown under standard conditions (˜100 uE/m²/s, 16 h photoperiod, 22° C. at day, 20° C. at night). Seeds were stratified for 3 days at 4° C. Seedlings were grown vertically (at a temperature of 22° C. at day 20° C. at night). Observations were taken on day 10 and day 14. Both seedling weight and root length at day 14 were analyzed as quantitative responses according to example 1M.

A list recombinant DNA constructs that improve early plant growth and development illustrated in Table 11.

TABLE 11 Root Root Seedling length length weight PEP at day 10 at day 14 at day 14 SEQ Construct Nomination Delta P- Delta P- Delta P- ID ID ID Orientation mean value mean value mean value 227 19774 CGPG3920 SENSE 0.488 0.097 0.307 0.100 0.550 0.163 229 19956 CGPG3972 SENSE 0.248 0.062 0.154 0.214 0.284 0.001 232 19973 CGPG4026 SENSE 0.326 0.047 0.066 0.577 0.444 0.012 302 70812 CGPG607 SENSE 0.704 0.037 0.546 0.031 0.740 0.033 236 70915 CGPG4058 SENSE 0.292 0.005 0.278 0.008 0.331 0.114 230 70948 CGPG3990 SENSE 0.255 0.269 0.093 0.229 0.375 0.026 234 70950 CGPG4052 SENSE 0.106 0.577 0.124 0.374 0.239 0.041 235 70962 CGPG4057 SENSE 0.171 0.075 0.144 0.226 0.283 0.015 241 70963 CGPG4121 SENSE 0.149 0.057 0.112 0.074 0.062 0.513 242 70994 CGPG4122 SENSE 0.198 0.063 0.121 0.114 0.240 0.124 244 70995 CGPG4154 SENSE 0.132 0.052 0.101 0.006 0.198 0.045 249 71328 CGPG4454 SENSE 0.209 0.136 0.251 0.122 0.445 0.093 268 72001 CGPG5221 SENSE 0.183 0.060 0.142 0.042 0.195 0.149 274 72753 CGPG5540 SENSE 0.113 0.045 0.053 0.348 0.081 0.730 282 73105 CGPG5656 SENSE 0.046 0.577 0.138 0.057 0.298 0.142 292 73136 CGPG5764 SENSE 0.212 0.077 0.133 0.027 0.330 0.037 265 73256 CGPG5194 SENSE 0.123 0.503 0.371 0.135 0.623 0.069 313 74420 CGPG6712 SENSE 0.249 0.085 0.093 0.358 0.363 0.042 316 74566 CGPG6796 SENSE 0.169 0.003 0.147 0.114 0.173 0.171 304 74662 CGPG6185 SENSE −0.205 0.337 0.009 0.878 −0.009 0.958 312 74688 CGPG6653 SENSE 0.221 0.083 0.059 0.358 0.295 0.163 329 74862 CGPG7308 SENSE 0.201 0.122 0.044 0.515 0.383 0.006 325 76178 CGPG7225 SENSE 0.217 0.018 0.093 0.364 0.240 0.234 322 77069 CGPG7163 SENSE / / / / 0.641 0.074 376 77124 CGPG9207 SENSE 0.337 0.121 0.256 0.045 0.762 0.034 371 77155 CGPG9170 SENSE 0.181 0.262 0.265 0.035 0.419 0.000 367 77164 CGPG9147 SENSE 0.589 0.068 0.369 0.066 0.899 0.051 370 77166 CGPG9163 SENSE 0.383 0.025 0.391 0.006 0.787 0.019 372 77180 CGPG9180 SENSE 0.442 0.058 0.267 0.031 0.402 0.339 362 77186 CGPG9133 SENSE 0.448 0.022 0.383 0.079 0.654 0.057 365 77187 CGPG9141 SENSE 0.453 0.043 0.227 0.127 0.660 0.013 375 77195 CGPG9205 SENSE 0.221 0.114 0.188 0.160 0.259 0.064 385 77209 CGPG9278 SENSE 0.360 0.092 0.181 0.203 0.594 0.047 382 77266 CGPG9259 SENSE / / / / 0.470 0.020 384 77268 CGPG9275 SENSE 0.353 0.111 0.170 0.244 0.386 0.041 378 77273 CGPG9220 SENSE 0.255 0.070 0.023 0.586 0.510 0.141 297 77312 CGPG5927 SENSE / / / / 1.006 0.065 393 77409 CGPG9345 SENSE 0.410 0.027 0.396 0.011 0.461 0.040 389 77430 CGPG9322 SENSE 0.209 0.183 0.211 0.247 0.399 0.092 390 77432 CGPG9335 SENSE 0.342 0.113 0.285 0.122 0.425 0.055 391 77433 CGPG9341 SENSE 0.204 0.120 0.222 0.041 0.382 0.085 392 77444 CGPG9344 SENSE 0.185 0.068 0.227 0.003 0.276 0.002 387 77451 CGPG9309 SENSE 0.266 0.097 0.208 0.042 0.014 0.981 388 77452 CGPG9311 SENSE 0.286 0.021 0.182 0.123 0.284 0.201 327 77536 CGPG7272 SENSE 0.121 0.389 0.147 0.026 0.384 0.027 342 77549 CGPG7933 SENSE 0.349 0.006 0.207 0.032 0.383 0.022 344 77568 CGPG8012 SENSE 0.312 0.020 0.157 0.015 0.359 0.071 351 77587 CGPG8107 SENSE 0.151 0.128 0.223 0.014 0.370 0.030 318 77618 CGPG6810 SENSE 0.179 0.018 0.104 0.276 0.154 0.388 334 77816 CGPG7529 SENSE 0.145 0.400 0.171 0.142 0.312 0.181 336 77821 CGPG7737 SENSE 0.114 0.406 0.126 0.208 0.311 0.079 343 77917 CGPG7986 SENSE 0.136 0.083 0.162 0.011 −0.242 0.458

If p<0.05 and delta or risk score mean >0, the transgenic plants showed statistically significant trait improvement as compared to the reference. If p<0.2 and delta or risk score mean >0, the transgenic plants showed a trend of trait improvement as compared to the reference.

Transgenic plants comprising recombinant DNA expressing a protein as set forth in SEQ ID NO: 252, 309, or 310 showed improved early plant growth and development evidenced by the second criteria as illustrated in Example 1L and 1M.

J. Late Plant Growth and Development Screen

This example sets forth a soil based phenotypic platform to identify genes that confer advantages in the processes of leaf development, flowering production and seed maturity to plants.

Arabidopsis plants were grown on a commercial potting mixture (Metro Mix 360, Scotts Co., Marysville, Ohio) consisting of 30-40% medium grade horticultural vermiculite, 35-55% sphagnum peat moss, 10-20% processed bark ash, 1-15% pine bark and a starter nutrient charge. Soil was supplemented with Osmocote time-release fertilizer at a rate of 30 mg/ft³. T2 seeds were imbibed in 1% agarose solution for 3 days at 4° C. and then sown at a density of −5 per 2½″ pot. Thirty-two pots were ordered in a 4 by 8 grid in standard greenhouse flat. Plants were grown in environmentally controlled rooms under a 16 h day length with an average light intensity of ˜200 μmoles/m²/s. Day and night temperature set points were 22° C. and 20° C., respectively. Humidity was maintained at 65%. Plants were watered by sub-irrigation every two days on average until mid-flowering, at which point the plants were watered daily until flowering was complete.

Application of the herbicide glufosinate was performed to select T2 individuals containing the target transgene. A single application of glufosinate was applied when the first true leaves were visible. Each pot was thinned to leave a single glufosinate-resistant seedling ˜3 days after the selection was applied.

The rosette radius was measured at day 25. The silique length was measured at day 40. The plant parts were harvested at day 49 for dry weight measurements if flowering production was stopped. Otherwise, the dry weights of rosette and silique were carried out at day 53. The seeds were harvested at day 58. All measurements were analyzed as quantitative responses according to example 1M.

A list of recombinant DNA constructs that improve late plant growth and development illustrated in Table 12.

TABLE 12 Rosette dry Rosette Seed net dry Silique dry Silique weight radius at weight weight length at PEP at day 53 day 25 at day 62 at day 53 day 40 SEQ Construct Delta P- Delta P- Delta P- Delta P- Delta P- ID ID mean value mean value mean value mean value mean value 394 12313 −0.075 0.489 0.654 0.000 0.157 0.174 −0.024 0.275 221 18258 −0.494 0.017 −0.183 0.083 0.533 0.006 −0.269 0.234 0.056 0.009 246 70657 0.373 0.040 0.187 0.037 0.355 0.081 −0.187 0.222 −0.003 0.972 247 70660 −0.010 0.936 −0.072 0.431 0.445 0.026 0.116 0.369 0.145 0.045 216 71538 −0.167 0.168 0.017 0.686 0.521 0.005 −0.196 0.113 0.023 0.557 269 72056 0.563 0.008 NA NA −0.251 0.159 −0.289 0.314 −0.046 0.314 279 73057 −0.266 0.035 0.167 0.114 0.396 0.034 −0.304 0.085 0.023 0.543 311 73568 −0.140 0.115 −0.085 0.340 0.553 0.019 0.344 0.010 0.040 0.458 222 19193 −0.296 0.110 0.081 0.385 1.076 0.011 NA NA 0.073 0.001 262 73242 0.159 0.132 0.133 0.050 1.074 0.009 0.652 0.002 0.045 0.048 309 73433 0.477 0.026 0.113 0.120 1.140 0.006 0.552 0.016 −0.009 0.872 316 74566 −0.387 0.023 0.104 0.047 1.213 0.003 −0.169 0.130 0.012 0.598 338 75654 0.604 0.012 −0.006 0.899 −1.193 0.004 −0.258 0.057 −0.004 0.962 361 77150 0.611 0.003 −0.098 0.115 −0.363 0.011 −0.398 0.203 0.001 0.990 327 77536 0.043 0.692 0.089 0.040 0.725 0.015 0.419 0.016 0.075 0.105 334 77816 −0.324 0.021 0.005 0.954 1.207 0.000 −0.038 0.679 0.039 0.378

If p<0.05 and delta or risk score mean >0, the transgenic plants showed statistically significant trait improvement as compared to the reference. If p<0.2 and delta or risk score mean >0, the transgenic plants showed a trend of trait improvement as compared to the reference.

K. Low Nitrogen Tolerance Screen

Under low nitrogen conditions, Arabidopsis seedlings become chlorotic and have less biomass. This example sets forth the limited nitrogen tolerance screen to identify Arabidopsis plants transformed with the gene of interest that are altered in their ability to accumulate biomass and/or retain chlorophyll under low nitrogen condition.

T2 seeds were plated on glufosinate selection plates containing 0.5×N-Free Hoagland's T 0.1 mM NH₄NO₃ T 0.1% sucrose T 1% phytagel media and grown under standard light and temperature conditions. At 12 days of growth, plants were scored for seedling status (i.e., viable or non-viable) and root length. After 21 days of growth, plants were scored for BASTA resistance, visual color, seedling weight, number of green leaves, number of rosette leaves, root length and formation of flowering buds. A photograph of each plant was also taken at this time point.

The seedling weight and root length were analyzed as quantitative responses according to example 1M. The number green leaves, the number of rosette leaves and the flowerbud formation were analyzed as qualitative responses according to example 1L. The leaf color raw data were collected on each plant as the percentages of five color elements (Green, DarkGreen, LightGreen, RedPurple, YellowChlorotic) using a computer imaging system. A statistical logistic regression model was developed to predict an overall value based on five colors for each plant.

A list of recombinant DNA constructs that improve low nitrogen availability tolerance in plants illustrated in Table 13.

TABLE 13 Leaf color PEP Root length Risk Rosette weight SEQ Construct Nomination Delta P- score P- Delta P- ID ID ID Orientation mean value mean value mean value 200 10422 CGPG117 ANTI- −0.435 0.008 1.311 0.021 0.050 0.396 SENSE 206 12602 CGPG170 SENSE −0.004 0.941 −0.052 0.895 0.115 0.048 202 13235 CGPG1288 SENSE −0.047 0.267 −0.085 0.864 0.128 0.068 203 13411 CGPG1301 SENSE 0.323 0.202 −3.233 0.126 0.174 0.044 201 13485 CGPG1226 ANTI- 0.311 0.096 1.162 0.038 0.241 0.019 SENSE 205 13846 CGPG1542 ANTI- −0.267 0.190 1.506 0.004 0.176 0.281 SENSE 212 16610 CGPG2499 SENSE −0.037 0.677 1.325 0.004 −0.004 0.918 211 17805 CGPG2457 ANTI- −0.348 0.016 3.523 0.067 −0.105 0.116 SENSE 218 18231 CGPG3274 SENSE −0.207 0.117 0.925 0.062 −0.093 0.094 224 18354 CGPG3534 SENSE 0.104 0.070 −0.407 0.124 0.189 0.048 252 70684 CGPG4588 SENSE 0.379 0.001 −3.676 0.003 0.328 0.030 223 71301 CGPG3528 SENSE −0.394 0.027 1.269 0.042 −0.128 0.026 250 71329 CGPG4456 SENSE −0.169 0.230 0.475 0.074 −0.365 0.337 258 72813 CGPG4977 SENSE −0.361 0.193 0.521 0.076 0.013 0.120 309 73433 CGPG6429 SENSE 0.129 0.201 −0.091 0.794 0.186 0.002 300 74349 CGPG5967 SENSE −0.139 0.019 0.592 0.080 0.045 0.080 313 74420 CGPG6712 SENSE 0.612 0.043 −3.086 0.063 0.173 0.002 298 75237 CGPG5941 SENSE −0.097 0.237 0.948 0.031 0.074 0.136 333 75379 CGPG7520 SENSE 0.000 0.998 0.529 0.048 −0.017 0.755 335 75434 CGPG7636 SENSE −0.336 0.039 1.208 0.076 −0.109 0.107 341 75622 CGPG7833 SENSE −0.503 0.001 1.776 0.007 0.096 0.497 339 75692 CGPG7823 SENSE −0.055 0.165 −0.377 0.328 0.132 0.083 301 76422 CGPG6040 SENSE 0.197 0.178 −0.263 0.734 0.162 0.094 359 76961 CGPG9080 SENSE −0.361 0.136 1.472 0.013 −0.190 0.066 369 77165 CGPG9155 SENSE 0.366 0.041 −1.982 0.143 0.253 0.099 303 77322 CGPG6178 SENSE / / 1.691 0.060 −0.185 0.052 207 16322 CGPG1828 SENSE / / −0.021 0.982 0.116 0.043 356 77832 CGPG8976 SENSE / / 2.472 0.011 −0.028 0.445

For leaf color and rosette weight, if p<0.05 and delta or risk score mean >0, the transgenic plants showed statistically significant trait improvement as compared to the reference. If p<0.2 and delta or risk score mean >0, the transgenic plants showed a trend of trait improvement as compared to the reference with p<0.2. For root length, if p<0.05, the transgenic plants showed statistically significant trait improvement as compared to the reference. If p<0.2, the transgenic plants showed a trend of trait improvement as compared to the reference.

Transgenic plants comprising recombinant DNA expressing a protein as set forth in SEQ ID NO: 198 or 327 showed improved tolerance to low nitrogen condition evidenced by the second criteria as illustrated in Example 1L and 1M.

L. Statistic Analysis for Qualitative Responses

A list of responses that were analyzed as qualitative responses illustrated in Table 14.

TABLE 14 categories (success response screen vs. failure) Wilting response Risk Soil drought tolerance non-wilted vs. wilted Score screen growth stage at day 14 heat stress tolerance 50% of plants reach screen stage1.03 vs. not growth stage at day 14 salt stress tolerance 50% of plants reach screen stage1.03 vs. not growth stage at day 14 PEG induced osmotic 50% of plants reach stress tolerance screen stage1.03 vs. not growth stage at day 7 cold germination 50% of plants reach tolerance screen stage 0.5 vs. not number of rosette leaves Shade tolerance screen 5 leaves appeared at day 23 vs. not Flower bud formation at Shade tolerance screen flower buds appear day 23 vs. not leaf angle at day 23 Shade tolerance screen >60 degree vs. <60 degree number of green leaves limited nitrogen 6 or 7 leaves appeared at day 21 tolerance screen vs. not number of rosette leaves limited nitrogen 6 or 7 leaves appeared at day 21 tolerance screen vs. not Flower bud formation at limited nitrogen flower buds appear day 21 tolerance screen vs. not Plants were grouped into transgenic and reference groups and were scored as success or failure according to Table 14. First, the risk (R) was calculated, which is the proportion of plants that were scored as of failure plants within the group. Then the relative risk (RR) was calculated as the ratio of R (transgenic) to R (reference). Risk score (RS) was calculated as −log₂ ^(RR). Two criteria were used to determine a transgenic with enhanced trait(s). Transgenic plants comprising recombinant DNA disclosed herein showed trait enhancement according to either or both of the two criteria.

For the first criteria, the risk scores from multiple events of the transgene of interest were evaluated for statistical significance by t-test using SAS statistical software (SAS 9, SAS/STAT User's Guide, SAS Institute Inc, Cary, N.C., USA). RS with a value greater than 0 indicates that the transgenic plants perform better than the reference. RS with a value less than 0 indicates that the transgenic plants perform worse than the reference. The RS with a value equal to 0 indicates that the performance of the transgenic plants and the reference don't show any difference. If p<0.05 and risk score mean >0, the transgenic plants showed statistically significant trait enhancement as compared to the reference. If p<0.2 and risk score mean >0, the transgenic plants showed a trend of trait enhancement as compared to the reference.

For the second criteria, the RS from each event was evaluated for statistical significance by t-test using SAS statistical software (SAS 9, SAS/STAT User's Guide, SAS Institute Inc, Cary, N.C., USA). The RS with a value greater than 0 indicates that the transgenic plants from this events perform better than the reference. The RS with a value less than 0 indicates that the transgenic plants from this event perform worse than the reference. The RS with a value equal to 0 indicates that the performance of the transgenic plants from this event and the reference don't show any difference. If p<0.05 and risk score mean >0, the transgenic plants from this event showed statistically significant trait enhancement as compared to the reference. If p<0.2 and risk score mean >0, the transgenic plants showed a trend of trait enhancement as compared to the reference. If two or more events of the transgene of interest showed improvement in the same response, the transgene was deemed to show trait enhancement.

M. Statistic Analysis for Quantitative Responses

A list of responses that were analyzed as quantitative responses illustrated in Table 15.

TABLE 15 response screen seed yield Soil drought stress tolerance screen seedling weight at day 14 heat stress tolerance screen root length at day 14 heat stress tolerance screen seedling weight at day 14 salt stress tolerance screen root length at day 14 salt stress tolerance screen root length at day 11 salt stress tolerance screen seedling weight at day 14 PEG induced osmotic stress tolerance screen root length at day 11 PEG induced osmotic stress tolerance screen root length at day 14 PEG induced osmotic stress tolerance screen rosette area at day 8 cold shock tolerance screen rosette area at day28 cold shock tolerance screen difference in rosette area cold shock tolerance screen from day 8 to day 28 root length at day 28 cold germination tolerance screen seedling weight at day 23 Shade tolerance screen petiole length at day 23 Shade tolerance screen root length at day 14 Early plant growth and development screen Seedling weight at day 14 Early plant growth and development screen Rosette dry weight at day 53 Late plant growth and development screen rosette radius at day 25 Late plant growth and development screen seed dry weight at day 58 Late plant growth and development screen silique dry weight at day 53 Late plant growth and development screen silique length at day 40 Late plant growth and development screen Seedling weight at day 21 Limited nitrogen tolerance screen Root length at day 21 Limited nitrogen tolerance screen

The measurements (M) of each plant were transformed by log₂ calculation. The Delta was calculated as log₂M(transgenic)-log₂M(reference). Two criteria were used to determine trait enhancement. A transgene of interest could show trait enhancement according to either or both of the two criteria. The measurements (M) of each plant were transformed by log₂ calculation. The Delta was calculated as log₂M(transgenic)-log₂M(reference). If the measured response was Petiole Length for the Low Light assay, Delta was subsequently multiplied by −1, to account for the fact that a shorter petiole length is considered an indication of trait enhancement.

For the first criteria, the Deltas from multiple events of the transgene of interest were evaluated for statistical significance by t-test using SAS statistical software (SAS 9, SAS/STAT User's Guide, SAS Institute Inc, Cary, N.C., USA). Delta with a value greater than 0 indicates that the transgenic plants perform better than the reference. Delta with a value less than 0 indicates that the transgenic plants perform worse than the reference. The Delta with a value equal to 0 indicates that the performance of the transgenic plants and the reference don't show any difference. If p<0.05 and risk score mean >0, the transgenic plants showed statistically significant trait enhancement as compared to the reference. If p<0.2 and risk score mean >0, the transgenic plants showed a trend of trait enhancement as compared to the reference.

For the second criteria, the delta from each event was evaluated for statistical significance by t-test using SAS statistical software (SAS 9, SAS/STAT User's Guide, SAS Institute Inc, Cary, N.C., USA). The Delta with a value greater than 0 indicates that the transgenic plants from this event performs better than the reference. The Delta with a value less than 0 indicates that the transgenic plants from this event perform worse than the reference. The Delta with a value equal to 0 indicates that the performance of the transgenic plants from this event and the reference don't show any difference. If p<0.05 and delta mean >0, the transgenic plants from this event showed statistically significant trait improvement as compared to the reference. If p<0.2 and delta mean >0, the transgenic plants showed a trend of trait enhancement as compared to the reference. If two or more events of the transgene of interest showed enhancement in the same response, the transgene was deemed to show trait improvement.

Example 2 Identification of Homologs

A BLAST searchable “All Protein Database” is constructed of known protein sequences using a proprietary sequence database and the National Center for Biotechnology Information (NCBI) non-redundant amino acid database (nr.aa). For each organism from which a DNA sequence provided herein was obtained, an “Organism Protein Database” is constructed of known protein sequences of the organism; the Organism Protein Database is a subset of the All Protein Database based on the NCBI taxonomy ID for the organism.

The All Protein Database is queried using amino acid sequence of cognate protein for gene DNA used in trait-improving recombinant DNA, i.e., sequences of SEQ ID NO: 198 through SEQ ID NO: 394 using “blastp” with E-value cutoff of 1e-8. Up to 1000 top hits were kept, and separated by organism names. For each organism other than that of the query sequence, a list is kept for hits from the query organism itself with a more significant E-value than the best hit of the organism. The list contains likely duplicated genes, and is referred to as the Core List. Another list was kept for all the hits from each organism, sorted by E-value, and referred to as the Hit List.

The Organism Protein Database is queried using amino acid sequences of SEQ ID NO: 198 through SEQ ID NO: 394 using “blastp” with E-value cutoff of 1e-4. Up to 1000 top hits are kept. A BLAST searchable database is constructed based on these hits, and is referred to as “SubDB”. SubDB was queried with each sequence in the Hit List using “blastp” with E-value cutoff of 1e-8. The hit with the best E-value is compared with the Core List from the corresponding organism. The hit is deemed a likely ortholog if it belongs to the Core List, otherwise it is deemed not a likely ortholog and there is no further search of sequences in the Hit List for the same organism. Likely orthologs from a large number of distinct organisms were identified and are reported by amino acid sequences of SEQ ID NO: 395 to SEQ ID NO: 19938. These orthologs are reported in Tables 2 as homologs to the proteins cognate to genes used in trait-improving recombinant DNA.

TABLE 2 SEQ ID NO: homolog SEQ ID NOs 198: 3331 2914 8408 6836 10845 14799 13562 8848 2509 13003 875 10945 4176 7318 4820 19156 19137 14942 17166 13809 3506 3410 1343 8019 7882 6490 3550 4999 3130 12635 8774 10864 17857 18089 16263 7295 10882 18252 3728 14833 11485 15463 18826 2924 18723 1446 2812 7847 3395 4209 15768 10679 18379 14040 3974 1087 11574 6329 5482 5609 6441 5537 17042 4716 4516 12643 11384 12736 8137 1344 19473 8452 4094 2460 18071 16891 5620 2364 7018 5200 11131 18133 12170 2731 1687 5014 18511 9343 19283 6682 8123 8124 10597 10622 10608 10600 16221 4280 11494 4867 9035 9507 7969 19159 19425 5248 17367 8135 8773 1287 2671 9365 18729 13828 8754 7170 14529 16604 8341 1135 6224 9739 11212 12962 4331 13555 14356 14357 14783 7509 2032 4692 5243 199: 15320 200: 7710 14519 16336 7019 7872 7719 18299 10453 4650 15008 10089 699 15645 10533 11751 12924 10526 5313 17532 8825 6941 7743 4155 14541 9797 16905 8304 12080 19803 3087 1397 7534 5547 2423 201: 11777 6444 1416 4353 15071 3413 14526 202: 19073 6651 15740 7399 15791 2977 7789 9248 14697 15923 12131 5470 965 11100 11069 12624 12892 3580 5270 18663 2916 1684 8107 4240 19240 18582 19054 15775 7301 17416 10566 16351 13846 16386 18431 4439 4056 8159 8292 12484 7316 3763 4783 18427 15501 16164 7762 4015 4556 18955 12061 8636 9665 9713 10212 17555 5330 12850 16436 19045 16034 16692 2571 11676 12666 11853 5348 8771 12218 3157 10749 203: 5608 2382 17993 16598 7505 6976 204: 11369 13527 10897 6893 6962 11592 837 5116 19669 1655 7244 2623 2591 16504 17797 4636 15133 4141 7112 7939 6898 15965 16706 16979 11784 16115 9620 15750 14652 3008 7838 14787 4563 1873 15811 7271 4317 16603 1715 14288 11341 6188 1546 5683 5225 3033 6069 11268 15981 15490 4181 8633 6058 7993 6763 9671 14892 17296 990 8184 2935 8536 11232 12598 12638 5112 10787 1107 13858 5864 16398 16380 17071 16520 1973 5455 13852 19228 1695 17402 2865 17120 2507 5601 14952 1462 6616 6973 3092 1382 7755 3870 9765 5711 1257 6119 15310 3792 15970 16032 14584 7282 15140 7273 3013 15249 13253 13241 2265 17700 18976 4009 17354 2223 17559 3151 6320 17010 8939 11258 734 573 15015 3050 16344 3931 11238 7691 11499 1112 4825 8344 13519 11669 5640 13588 5473 15108 7210 17617 5994 5980 16627 17906 5876 6079 18894 6956 12887 14694 10968 1061 2778 6096 10570 680 974 3120 16300 5885 7626 13881 4244 4233 15039 4486 3103 3115 7654 11890 12359 10800 9409 12657 8977 9182 17818 2942 9780 9899 18496 4118 18188 10437 13907 17312 15730 13906 2603 12770 4837 12953 16663 9237 9546 16293 18238 6022 13908 10266 9180 5538 15536 10370 15958 5217 8154 13095 15972 14841 15262 12702 10766 15960 18726 19395 10345 5964 13064 3946 17225 17262 17220 17266 17224 17252 5465 5495 12800 12814 12758 3021 12400 12492 16461 13638 5616 17449 8397 17258 5669 5675 6852 9796 17601 17569 9775 17664 205: 14023 6117 12539 6696 18601 1463 19893 6621 9577 9166 17890 17889 528 5413 18313 2784 8102 3988 890 19106 571 6791 2402 912 206: 17912 8799 6566 12137 4356 5803 5253 6078 9123 17523 12392 17806 6917 19039 7350 17767 11311 17082 15535 19392 11233 470 8318 14963 16736 8867 14135 5246 4674 2124 9901 13302 15459 18997 2954 1026 667 7561 16841 8092 9084 4065 1836 13713 13697 17640 7422 19580 4565 15193 526 13108 7065 11703 11362 3633 19430 6221 17433 15616 10082 10189 3089 15238 6633 8106 16180 15317 16364 207: 14575 8831 11211 14985 17619 18829 2521 5219 15413 15835 1204 6569 4661 4457 6066 14578 932 13694 7541 4813 15707 19563 1326 16107 5009 7518 14825 13777 6175 15925 13617 5765 14838 9091 10776 15679 5909 19373 756 208: 19581 18655 16310 7497 6382 8261 936 7358 8883 4866 2146 8601 2932 1626 10354 15192 11509 9909 13503 488 17477 14483 16941 2824 1072 4989 14179 4883 13751 209: 3493 16588 7031 5155 11150 17294 5241 10621 5786 5591 16509 2021 1324 12454 15061 1670 15143 16506 17002 8741 18091 4013 5959 19370 4239 12885 12174 3709 3431 9596 3378 1018 5963 13212 3150 2952 1619 19861 19114 15446 19582 12663 19603 210: 17777 843 11020 1337 18495 1493 6396 15336 14087 2568 4517 1157 10741 4553 14999 425 4258 17388 5327 748 11691 10174 17805 16628 10117 7980 8902 211: 9637 17261 13232 7387 10506 19067 12021 872 11620 13603 841 1450 2848 16911 212: 14398 1558 18339 13428 13431 4182 16260 10619 6127 12005 7704 5064 13072 4395 9032 19907 9588 478 12782 9917 13100 5202 10562 2044 4452 10775 3543 14840 14071 14020 10406 14218 1797 16955 3879 10312 12432 14824 7082 14554 15744 1508 15866 16232 6797 7077 5816 3597 3596 3311 15022 881 2638 906 12037 12039 3458 2270 8812 3217 9466 18102 610 9042 2431 18349 16650 14418 2251 4235 6826 1244 16651 13004 7852 14435 9553 9262 15603 4340 9235 1413 10538 8296 17223 16048 16071 2120 19847 10420 11239 18968 2995 7551 15854 12345 5685 18180 2381 4814 2159 16862 8677 13466 11435 6649 6015 8097 11230 16025 9440 1353 16151 16249 15930 11226 9398 2870 12373 15043 11765 10145 11071 285 9270 9537 9095 2350 13362 5496 7924 11756 2624 7967 6715 15074 17068 9275 10177 11007 14857 8951 12182 11643 13590 7684 1740 929 910 9093 10018 13344 16978 17544 6305 11378 1393 14108 15937 16631 11803 13126 11969 18524 9644 8425 8421 8420 10755 10757 16696 15994 1143 3549 5868 5126 7506 12547 12669 19032 4707 4710 4708 4732 4736 4733 8772 4730 4711 3385 1831 2528 2304 15098 13223 15197 11673 4386 11884 2336 14693 9118 9633 14548 12725 2399 18359 11011 14692 5652 5434 9755 9513 2681 4364 12903 19777 13525 4326 6859 6088 11611 3068 13940 3578 14440 8582 9826 19618 18012 17233 10057 18222 15021 9371 5985 14888 14001 1279 7826 14232 18172 7425 11502 15565 17431 8317 10294 17742 5564 5531 18144 7400 10835 14389 13461 13857 7113 16444 4722 2115 7276 18254 1237 5256 13951 2920 16719 1045 15371 19022 684 8004 2781 19811 19766 3279 18712 4940 14312 6426 10076 15772 7906 19836 11219 18985 2673 3933 14263 11860 1907 4672 15599 13737 4944 2252 15415 11543 3433 7746 17114 4115 4881 9445 14696 9525 14484 16112 5392 10428 1400 9349 7616 18930 2533 12585 10497 10236 18415 4874 19793 4633 12544 1995 17255 6130 10439 15261 6768 15985 17752 8504 12863 7487 14875 12020 2036 18127 9264 3628 5019 7498 15865 1448 9636 2840 3161 6207 2644 1059 2065 10031 2171 16927 4747 18701 19271 4174 1389 9893 12527 9018 10035 5039 10698 13943 7894 12671 5651 19586 12810 1254 3519 16114 4286 13948 14493 8294 14606 2586 14069 7712 16988 737 16797 16826 18966 5145 17610 6304 8895 7744 9280 9284 10647 16558 213: 12662 5295 19093 10971 15359 1163 18513 889 2151 3536 7105 3292 8466 18205 3579 1504 19486 13768 18047 6871 7174 11029 11505 17782 5697 18822 4892 12656 1284 3062 4697 5993 18343 18790 3057 7014 12265 10981 15204 11631 17704 17941 10633 19776 933 10363 8446 17800 9822 2953 13693 19862 14551 15692 17673 6081 5049 12581 6910 214: 15345 19484 4871 3871 6695 3993 11459 9199 15899 13821 14206 19489 13765 712 19072 11649 215: 1607 15669 13168 19044 790 9515 4511 13776 11573 13000 216: 4929 17143 10058 17506 15064 3332 5268 6968 16252 15949 6180 3000 14937 18454 13071 217: 6807 19716 480 14021 4179 12236 6337 12237 6338 6336 12234 4815 2261 18555 4706 7836 9176 18851 3443 9382 9439 8870 16691 16646 11516 11500 11515 11498 16670 16660 16683 11513 11496 16668 16664 11519 16686 11479 11481 7146 16643 16659 4850 17951 7144 4824 7163 4851 11234 4860 17956 7160 17977 4827 4826 4859 7147 11475 7165 17928 17958 4853 11473 7143 11477 5129 18893 18914 17526 17529 17548 17547 17549 18917 13686 19089 13683 4938 16999 13666 4494 12074 17097 12093 16874 12095 15878 17159 17154 17156 17153 5681 15126 10052 19712 4288 15399 742 11607 15654 7006 8041 715 2713 11522 7695 3581 7865 3776 15738 19821 17259 3948 13388 17427 15523 14216 8711 15423 19060 16814 14298 18720 7885 3967 9710 3568 3573 3575 8503 15486 12932 7870 989 3595 17756 9202 520 11205 14771 3574 2443 8036 19537 14641 6739 15375 2400 6753 3336 218: 13099 6217 4454 4699 11292 4702 1554 6467 15339 15722 11035 12964 16317 13914 17486 18809 14651 4189 9581 14957 2871 1561 17618 3835 14955 3868 9158 13663 10042 7575 19824 6200 10522 3600 4064 14590 12731 7201 19572 9060 4035 6255 3457 11320 7986 8818 219: 19325 10450 4487 12312 2564 10096 5641 1947 4367 6964 18031 1012 11041 15078 12255 5761 5061 9832 16834 18348 220: 8238 19207 18657 1358 2734 9320 13526 12631 17660 16548 15528 18501 18049 15274 19470 221: 2724 17089 9639 9208 17722 6817 11848 18904 13673 2347 11585 3054 7486 6056 1290 14932 14930 12636 14230 1572 15708 18912 18177 1699 10460 2090 5583 18802 606 5929 5041 19148 14224 9144 2643 17144 14142 14674 16275 222: 14820 18626 11591 649 3005 3037 5453 5430 14248 2371 4626 11661 9784 10560 16219 11699 223: 17739 13288 4139 15466 19773 753 1277 14333 4211 14510 662 7930 18703 3268 19400 17533 2302 9078 224: 13859 17816 12692 10417 7051 5444 12975 1611 7050 17105 5023 3397 13149 14751 10564 7331 7329 11868 9210 9211 18860 18879 14568 17920 14497 12723 19352 10587 225: 11265 17061 11261 17058 11267 17064 8014 8033 8018 508 8035 8038 8013 8017 3554 13411 12545 12528 11997 12566 11922 12520 11958 12525 12522 12553 11961 11979 5030 3464 3910 18901 14601 14600 840 15664 15668 15121 15142 15119 15686 15690 15657 15688 15146 15164 15716 15148 15656 11191 13623 12732 5050 5029 5011 5055 5035 5036 5032 5081 5058 5013 5078 5026 5059 5077 5008 5053 5076 11939 11976 12541 2898 17207 5179 5115 13674 13423 16078 9689 11174 5252 17279 17277 11177 1867 1865 1823 1844 1842 1793 1824 1820 10671 3139 1123 5805 12934 12755 7504 1091 17904 17909 17926 17902 17938 17907 17929 10598 17935 17933 5298 17980 10601 5383 14050 6585 11379 16583 17978 17814 4838 17812 2633 3257 3189 774 12008 9988 14268 18392 12567 14920 12980 11959 7682 7685 13235 4127 3740 3766 3770 3769 3795 14966 457 8521 19464 4847 13386 5024 13387 13401 5957 6571 7932 8369 10496 10492 10490 10495 10517 7943 4958 4956 4923 4986 4961 4951 5007 4937 4934 4984 4933 4924 4921 4936 4979 4932 4981 4988 18200 18196 7176 4739 5479 8351 7177 12886 9521 6464 17681 14504 907 16793 1869 14509 8882 19727 8798 1197 3419 14168 12846 15739 1115 14165 19715 492 8873 1119 497 1194 3251 1116 8876 13982 12860 14430 451 14969 6102 4358 2650 2649 11790 12925 5882 19696 9551 3867 19796 18933 18929 833 4908 14217 4852 4858 16064 14208 10736 4886 14214 16062 14172 10735 14210 10720 1885 14187 14190 14184 14207 14703 14707 14709 14706 14390 7658 7659 7661 2582 13794 5204 17305 16541 8914 16959 17283 9043 3924 2696 8656 6648 2675 18504 17311 6052 9425 9406 9410 9411 6051 9452 6033 15964 6267 6031 8489 8463 8865 9427 6268 8490 8493 15602 15621 14616 15649 15652 15606 11723 17247 9423 15595 15624 16148 2240 7428 11722 11363 2225 2196 5480 15579 15626 16142 7413 5483 11741 16145 16162 11725 5481 18072 10973 12451 12340 15511 12185 4348 12754 8722 14544 6171 10732 9492 19795 12165 15082 6617 9613 739 19520 2497 14005 11128 18795 14331 14329 14334 16439 12070 4053 3942 2387 2391 2394 755 754 2950 752 2377 19819 17119 7118 1360 2273 11458 13758 11215 11274 11229 13178 5110 16239 12884 18765 16637 19693 16831 17824 10810 15395 15420 17470 10005 16397 16394 14711 14728 15503 4401 801 12244 12292 12308 12219 9033 16763 14392 16804 16785 9566 16806 14409 14391 16762 9027 9568 9029 16788 9009 16780 14410 16781 16782 16802 3330 3357 3326 9001 3358 5789 14387 14412 3360 3356 1643 1805 14061 15787 5632 226: 19514 17912 10095 10091 10252 15927 5360 18385 12313 19719 12823 5182 1491 8631 4666 2562 9358 10461 3952 19610 9255 7782 16516 5161 6343 4894 5577 16734 1381 16378 14552 12707 11907 7651 10404 17767 2417 11311 17082 17741 11253 11254 7473 13718 6269 3242 7085 5224 8787 16268 19692 10769 8867 1434 16082 19530 14789 13615 18997 2954 9175 8092 9082 1813 1815 1817 639 3883 15427 2035 4435 3601 414 7005 8000 1812 1131 1455 6221 11231 10817 15601 16270 6118 2193 17500 14186 9141 3449 3089 13886 15786 15238 2001 3945 14973 16180 15317 18083 18695 227: 17372 8093 5527 2439 13784 14768 10702 11535 7750 761 18735 5946 17426 17988 10652 2928 18398 2009 9559 5094 12948 8522 1305 17785 5852 228: 2790 5299 15433 5501 18128 16466 14960 6564 8782 4397 19662 16008 10829 19118 18086 3428 17943 10935 15806 8101 1184 4968 6059 10388 229: 7539 3499 12437 19471 14158 11575 6399 15966 17187 13512 7688 2131 10858 4169 19567 7834 7585 14073 3954 1991 12940 7756 8167 18994 9449 15859 4341 17025 14373 12191 12982 983 13764 19774 16247 12665 12664 2619 18875 7515 11785 2849 10913 10717 4653 11887 905 1641 3563 6183 16765 17773 4166 4694 14328 11986 12084 1625 12695 9681 14082 17341 15047 17498 3530 3531 7241 7972 4790 15643 14365 230: 12430 9914 10470 14976 6139 18895 8072 18544 7257 6437 15066 231: 7185 13859 12150 12692 5555 6319 14735 16754 3121 2208 14915 18531 2991 7950 2419 4378 18922 4377 18705 17105 5023 3397 2203 5350 895 18953 5090 9210 9211 14568 19723 12433 3877 17892 19476 11685 9653 9066 16392 7989 5056 6545 19418 232: 10845 14019 10546 14942 7302 8774 13992 10864 7129 12564 10723 2440 19878 3172 18252 12532 9942 2129 18826 18723 17324 19791 8580 3395 5915 9604 7572 16359 8047 3408 5482 12915 9969 16513 1344 16044 19210 18748 11940 14402 19311 5620 2364 19283 5249 3646 17251 17844 14459 5315 9268 16190 19143 15124 418 3076 16204 17414 18692 13164 8191 10636 13894 3423 2842 1173 7663 13250 18208 14090 19680 15815 4805 10584 1287 15495 9256 2229 10166 19534 11163 2292 2687 19082 9365 2465 5381 18660 3073 9545 17750 233: 19344 12031 1229 15123 14123 19501 7767 5944 11173 4596 11614 10841 14129 2393 7360 621 14382 11612 8136 14668 7577 1652 2491 3392 2160 4767 17696 6615 12377 3471 2985 17194 17177 10988 5084 9695 16519 11445 3213 10846 16453 19346 18274 17478 17798 15663 6823 5131 14272 3777 19911 19724 6132 19722 14803 17148 1605 11787 14359 15796 645 10159 13264 12931 6357 4621 16774 18913 5746 7161 234: 11849 7119 1272 13625 6244 7780 7229 640 15779 14967 5388 14480 1349 17525 12768 10327 8976 5113 5895 15006 4428 16619 4613 3393 18954 4451 3799 17712 12488 15863 18697 18482 7844 235: 8664 9584 8904 18497 19265 8237 2617 7729 17728 8659 13842 11016 9197 10827 5351 3224 7379 236: 12797 7459 12082 1618 7740 16856 7623 8994 7602 19549 12596 7644 18396 13883 7266 18758 3328 18998 1660 12434 2139 19899 12252 5883 3266 19587 11474 5870 5443 237: 14746 16460 7754 9978 16450 18053 18050 18466 8866 10337 8245 1604 8568 13606 19627 18283 15611 10648 13871 9910 10297 3025 18486 8908 14474 11654 2349 10867 14488 13901 7336 11511 14836 1906 13400 5568 19527 4351 18319 6689 10519 15977 15760 7605 16652 14204 14665 727 9225 12804 2776 11108 14258 1882 4357 1972 8324 6435 18239 6728 18983 9555 13551 10797 18458 4622 14064 2771 1818 18410 13553 15912 10854 9475 10873 15530 14816 6283 703 8105 16216 7038 957 11908 13853 10718 11237 5623 10262 12248 6541 7053 4480 14011 17142 6691 7795 17981 2822 1473 3561 4545 13347 1348 11256 6511 11356 13955 6699 6555 6559 3185 16166 17894 9307 6011 10635 1391 13177 16458 702 18335 18740 16787 4175 10950 3412 4193 951 13870 19183 18644 947 16280 6184 13111 3667 10338 4125 14782 18479 18004 19084 12471 16645 866 1895 10049 11122 12521 7132 2660 12759 14534 18429 15615 17611 9347 12704 1525 7492 3451 18373 17125 5972 9777 16039 12350 4073 8962 17221 1961 1958 11156 12899 3908 7429 10977 15040 16234 17004 17293 13355 4977 260 10645 14133 9251 12220 12633 7087 9535 8894 11452 2346 6125 2062 6710 13299 12947 12943 4422 5817 13502 14378 13504 14379 4770 1451 19815 12842 4405 13837 9184 10051 10999 9565 12305 11330 16546 17073 1402 5263 7189 6160 2372 6567 1248 3858 14455 1766 2516 1656 14550 16864 14989 14368 15573 9312 7786 13516 2969 1952 2541 11207 10625 9198 19674 10334 18065 3495 8519 12201 3921 13249 18592 857 17545 11757 15455 2164 789 4350 3117 8649 11708 11463 238: 9324 12427 663 6064 9836 19556 9348 1900 17219 13657 7086 7381 6006 7357 10469 6466 8472 10588 17047 609 1637 10699 6450 14299 15101 10536 18759 2378 18013 9269 3343 12489 11305 18178 2269 16890 10629 18665 15962 9375 9437 16312 6501 11737 6339 10847 5290 4667 5522 18388 1804 11566 2369 15872 12465 7687 6209 11354 15667 15938 18971 18258 3774 239: 13622 9907 10802 1635 19607 8218 11862 18989 19820 1430 15998 4855 18040 3455 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5500 4771 6745 5378 3236 1210 11603 19433 19342 15300 17953 11454 9261 12841 11591 5415 5453 5430 8278 14564 6761 17927 18569 14781 10539 6214 3620 3528 17899 8748 12221 5504 5502 9489 6425 8958 382: 19132 10614 8139 10836 16040 11545 7879 2100 1703 14547 383: 1189 16611 3586 4641 19633 17705 17150 8849 3552 7294 2029 6153 18596 384: 741 1633 5923 8765 16673 14609 3698 6449 2951 2093 11563 614 14593 14762 6397 7731 14290 17038 4311 14640 385: 11357 13113 2101 9359 2343 5986 9703 12857 8854 17190 4889 13808 12572 3386 7845 17983 4794 386: 1016 16046 10106 6272 16847 10542 15062 5576 4893 5448 15407 7831 16578 18278 2702 7215 9961 6250 3252 18599 16433 3226 17007 387: 7223 8111 7867 1322 9479 3291 18674 5132 12538 3515 18852 16518 14682 5419 9781 388: 13217 19387 1319 13219 1333 2384 4322 7600 13280 5924 13688 2181 16877 16122 5091 927 6100 3686 389: 11269 4678 13073 1755 1754 8654 3662 17371 3891 3973 2187 15246 18462 4802 3390 619 643 13183 4014 1542 10000 19057 6229 1262 14305 14256 17590 5708 4877 3775 8462 390: 17182 3673 6158 5731 8195 15335 7264 7267 5346 10728 11675 2337 19110 7961 8491 12046 10224 6729 18918 18228 3153 8723 3309 3313 6222 15956 4632 12325 6835 6387 2622 4496 9897 10860 8044 10003 13262 13236 6403 5331 4640 1303 4602 1304 18435 16053 18480 17646 17644 17675 16363 4781 3269 8610 10852 3582 5717 9242 9340 9346 13271 1345 3780 11086 14006 2264 5062 5146 16786 891 1282 11639 2472 9621 19732 15665 10462 16384 18138 9510 19888 19119 4931 18991 13945 18937 13580 18401 13863 1936 4639 10245 760 13679 14995 17389 5259 10385 9763 15381 10901 10886 10888 12415 15437 8840 7871 4684 12209 17107 11203 16435 8624 13410 8530 10422 11650 15110 13639 12675 13689 19825 4662 13277 5625 9279 3369 5976 1377 2144 4086 18044 16889 6767 18418 11859 8188 6500 16529 16528 13234 14259 18077 11012 10071 11825 16181 6479 3800 9154 19377 924 923 7361 4574 14380 13895 17168 14901 17969 13735 17599 10270 4099 18973 3523 14337 8378 4490 18286 5203 2114 13850 9109 9096 7060 4161 6561 1211 9640 13790 19180 14796 5771 15355 14027 6282 12681 16502 4842 4262 4930 15500 16490 14450 18361 15929 1171 7624 13836 15083 19541 1490 8022 9426 19755 13700 10435 8346 13719 8681 19368 10906 19116 1509 3865 12853 3125 5889 8316 18315 9077 436 1977 8354 5002 5025 16553 5171 17152 9352 17908 9960 9963 4907 2079 14164 3744 3102 18375 4128 4129 14794 391: 5830 10547 4780 6670 1560 15309 12977 11581 15243 10500 7299 19550 4614 11584 18988 17503 13880 18201 7124 6036 17359 15414 19074 13696 4180 810 12806 13939 1259 2434 12029 5666 8718 751 18491 15717 1795 4475 1738 627 5732 18253 8660 11107 16309 12868 4299 14698 14679 14502 8248 11990 9758 16167 9385 10043 10430 16906 12099 5791 16810 13759 8562 2921 8889 10315 14077 2682 18457 14406 12241 10194 5832 13548 15304 4676 392: 18452 9432 5303 14233 9970 7637 14980 15265 7581 3016 10412 19809 393: 19384 19382 682 6463 1021 6039 6041 6057 7898 7896 13532 13533 8037 9356 16887 16866 7150 7148 11880 11903 7084 7083 3381 13816 13817 12463 4530 2311 2312 10624 10623 15054 2256 2257 14016 14018 8801 6140 6142 8800 6138 8357 8375 8360 3666 3668 11044 11022 9473 9494 5001 5000 4831 4836 18327 19232 5812 5815 9999 7941 7942 749 3079 7426 7451 8001 7999 8005 19635 4051 15885 12205 2860 786 784 15576 15575 12310 12307 2306 3461 7640 10242 394: 12840 13577 8132 1535 5905 3211 3797 12558 6843 17949 15017 9231 1310 17041 6367 18764 8461 15888 6866 6864 7983 8980 10079 15489 11102 9933 7325 1749 8441 7720 17727

Example 3 Consensus sequence build

ClustalW program is selected for multiple sequence alignments of an amino acid sequence of SEQ ID NO: 198 and its homologs, through SEQ ID NO: 394 and its homologs. Three major factors affecting the sequence alignments dramatically are (1) protein weight matrices; (2) gap open penalty; (3) gap extension penalty. Protein weight matrices available for ClustalW program include Blosum, Pam and Gonnet series. Those parameters with gap open penalty and gap extension penalty were extensively tested. On the basis of the test results, Blosum weight matrix, gap open penalty of 10 and gap extension penalty of 1 were chosen for multiple sequence alignment. The consensus sequence of SEQ ID NO: 227 and its 25 homologs were derived according to the procedure described above and is displayed in FIG. 1.

Example 4 Identification of Amino Acid Domain by Pfam Analysis

This example illustrates the identification of amino acid domain by Pfam analysis.

The amino acid sequence of the expressed proteins that were shown to be associated with an enhanced trait were analyzed for Pfam protein family against the current Pfam collection of multiple sequence alignments and hidden Markov models using the HMMER software in the appended computer listing. The Pfam protein families for the proteins of SEQ ID NO: 198 through 394 are shown in Table 16. The Hidden Markov model databases for the identified patent families are also in the appended computer listing allowing identification of other homologous proteins and their cognate encoding DNA to enable the full breadth of the invention for a person of ordinary skill in the art. Certain proteins are identified by a single Pfam domain and others by multiple Pfam domains. For instance, the protein with amino acids of SEQ ID NO: 207 is characterized by two Pfam domains, i.e. “TFIIS_M” and “TFIIS_C”. See also the protein with amino acids of SEQ ID NO: 213 which is characterized by three copies of the Pfam domain “WD40”. In Table 16 “score” is the gathering score for the Hidden Markov Model of the domain which exceeds the gathering cutoff reported in Table 17.

TABLE 16 PEP SEQ Pfam domain ID NO GENE ID name begin stop score E-value 198 CGPG106 CMAS 69 306 −149.8 5.00E−05 198 CGPG106 Methyltransf_11 131 229 109.1 1.20E−29 198 CGPG106 Methyltransf_12 131 227 54.6 3.10E−13 200 CGPG117 SNF5 14 252 66.9 5.90E−17 202 CGPG1288 Cupin_3 57 131 130.8 3.40E−36 204 CGPG1458 Phosphorylase 180 958 1454.7 0 205 CGPG1542 LMBR1 11 488 715.1 4.40E−212 206 CGPG170 Metallophos 54 249 140.4 4.50E−39 207 CGPG1828 TFIIS_M 206 327 203.5 4.50E−58 207 CGPG1828 TFIIS_C 338 376 83.3 7.00E−22 209 CGPG2217 ORMDL 14 156 356.8 3.20E−104 210 CGPG2292 Tim17 52 186 16.1 4.20E−05 210 CGPG2292 SAM_1 192 253 13.8 0.019 212 CGPG2499 Glycolytic 4 339 588.8 4.50E−174 213 CGPG2653 WD40 135 172 40.3 6.20E−09 213 CGPG2653 WD40 235 271 40.7 4.60E−09 213 CGPG2653 WD40 276 313 38 3.00E−08 214 CGPG2813 HLH 204 251 34.1 4.60E−07 215 CGPG3002 eIF-48 5 451 −50.3 1.90E−10 216 CGPG3154 C2 14 102 101.8 1.80E−27 216 CGPG3154 C2 198 272 57.8 3.20E−14 217 CGPG3235 B_lectin 66 176 156.8 5.30E−44 217 CGPG3235 S_locus_glycop 189 315 227 3.90E−65 217 CGPG3235 PAN_2 332 402 103.9 4.20E−28 217 CGPG3235 Pkinase_Tyr 488 757 75.6 2.10E−20 217 CGPG3235 Pkinase 488 757 109 1.30E−29 218 CGPG3274 Allene_ox_cyc 74 253 401.6 1.10E−117 219 CGPG3275 Auxin_inducible 50 155 135.6 1.30E−37 220 CGPG3363 zf-AN1 21 58 10.5 0.0033 220 CGPG3363 zf-AN1 103 148 12.4 0.0019 221 CGPG3367 TFIIA 9 375 556 3.40E−164 222 CGPG3375 Tryp_alpha_amyl 33 111 55.2 1.90E−13 224 CGPG3534 Ank 42 74 13.6 0.31 224 CGPG3534 Ank 75 107 36.8 6.90E−08 224 CGPG3534 Ank 108 140 1.6 17 224 CGPG3534 Pkinase_Tyr 160 413 202.4 9.90E−58 224 CGPG3534 Pkinase 160 413 198.5 1.40E−56 225 CGPG3638 Ribonuclease_T2 30 219 367.3 2.20E−107 226 CGPG3918 Metallophos 60 255 159.1 1.10E−44 229 CGPG3972 14-3-3 8 243 501.1 1.20E−147 230 CGPG3990 zf-C3HC4 259 299 47.3 4.60E−11 231 CGPG3994 Pkinase 554 802 207 4.10E−59 231 CGPG3994 Pkinase_Tyr 554 802 233 5.90E−67 232 CGPG4026 CMAS 59 320 −174.5 0.003 232 CGPG4026 Methyltransf_11 122 220 96.3 8.10E−26 232 CGPG4026 Methyltransf_12 122 218 37 5.90E−08 232 CGPG4026 Sterol_MT_C 229 358 301.1 1.80E−87 233 CGPG4048 Pkinase 21 275 342.1 8.80E−100 233 CGPG4048 Pkinase_Tyr 21 275 65.1 1.20E−19 233 CGPG4048 NAF 304 364 104.5 2.80E−28 234 CGPG4052 DUF298 127 242 222.4 9.20E−64 236 CGPG4058 Asp 99 437 −128.4 4.60E−06 237 CGPG4069 adh_short 31 218 −16.7 4.80E−05 239 CGPG4088 SWIB 324 399 96.4 7.80E−26 241 CGPG4121 Cyclin_N 58 190 133.7 4.80E−37 241 CGPG4121 Cyclin_C 192 314 44.3 3.70E−10 242 CGPG4122 zf-C3HC4 100 141 27.6 4.10E−05 243 CGPG4140 p450 48 484 140.8 3.30E−39 244 CGPG4154 zf-CCCH 90 115 22.6 0.0012 245 CGPG4311 FBPase 70 395 347.9 1.50E−101 247 CGPG4369 BolA 10 79 92.1 1.60E−24 248 CGPG442 2-Hacid_dh 85 394 140.9 3.20E−39 248 CGPG442 2-Hacid_dh_C 187 362 293 5.20E−85 248 CGPG442 ACT 551 621 39.9 8.10E−09 249 CGPG4454 p450 45 447 −35.5 2.10E−09 250 CGPG4456 p450 75 502 103.6 5.30E−28 252 CGPG4588 Auxin_inducible 1 102 151.5 2.00E−42 253 CGPG4765 DUF868 28 304 175.5 1.20E−49 255 CGPG4912 WD40 243 281 31 3.80E−06 256 CGPG4926 WD40 42 79 22.8 0.0012 256 CGPG4926 WD40 126 163 25.8 0.00014 257 CGPG4967 Asp 71 424 −94.7 4.70E−08 258 CGPG4977 Usp 3 157 85.3 1.80E−22 259 CGPG5001 adh_short 5 181 6.4 1.40E−06 260 CGPG5025 adh_short 30 212 5.7 1.50E−06 261 CGPG5041 DUF26 77 132 84.3 3.40E−22 261 CGPG5041 DUF26 188 242 100.7 4.00E−27 261 CGPG5041 Pkinase 333 558 60.3 5.70E−15 262 CGPG5116 ArfGap 15 137 174.1 3.20E−49 262 CGPG5116 C2 182 261 101.4 2.40E−27 263 CGPG5144 p450 61 535 157.9 2.40E−44 264 CGPG5171 p450 35 496 137 4.60E−38 265 CGPG5194 DUF1191 25 308 616.2 2.60E−182 268 CGPG5221 p450 30 499 135.4 1.40E−37 269 CGPG5269 PCl 297 401 108.9 1.30E−29 270 CGPG5404 Peptidase_S10 68 480 683.3 1.70E−202 271 CGPG5432 MtN3_slv 12 99 145.4 1.40E−40 271 CGPG5432 MtN3_slv 133 219 140.4 4.40E−39 272 CGPG5518 Ribosomal_S8e 1 238 325.9 6.30E−95 273 CGPG5535 WD40 335 371 28.8 1.80E−05 273 CGPG5535 WD40 434 471 37.1 5.50E−08 273 CGPG5535 WD40 476 513 47.2 5.20E−11 273 CGPG5535 WD40 518 555 35.6 1.60E−07 273 CGPG5535 WD40 567 604 36.1 1.10E−07 273 CGPG5535 WD40 621 658 34.9 2.50E−07 273 CGPG5535 WD40 663 706 35.5 1.60E−07 274 CGPG5540 ESCRT-III 21 207 265.7 8.40E−77 275 CGPG5568 AA_permease 69 521 −67.1 0.00013 276 CGPG5577 SMC_N 21 1049 −2.8 1.50E−11 277 CGPG5587 Thioredoxin 78 199 0.1 0.00015 278 CGPG5594 Histone 27 100 99.6 8.80E−27 279 CGPG5633 PGAM 91 277 153.2 6.30E−43 280 CGPG5640 Aminotran_1_2 30 385 362.1 8.00E−106 281 CGPG5646 iPGM_N 2 363 876.8 9.10E−261 281 CGPG5646 Metalloenzyme 373 488 173.3 5.40E−49 282 CGPG5656 Gln-synt_N 17 99 85.7 1.30E−22 282 CGPG5656 Gln-synt_C 105 358 528.1 8.50E−156 283 CGPG5659 Aminotran_3 35 361 321.7 1.20E−93 284 CGPG5661 PK 1 345 702 3.80E−208 284 CGPG5661 PK_C 355 469 115.4 1.50E−31 285 CGPG5684 Glycolytic 4 339 578.6 5.60E−171 286 CGPG5694 TIM 4 246 465.9 4.50E−137 287 CGPG5704 NDK 2 136 329 7.30E−96 288 CGPG5714 NDK 4 138 266.4 5.20E−77 289 CGPG5721 Rib_5-P_isom_A 47 215 354.4 1.70E−103 290 CGPG5728 zf-CCCH 80 106 39.3 1.20E−08 291 CGPG5757 Sad1_UNC 203 330 195.6 1.00E−55 292 CGPG5764 Actin 11 140 36.4 6.60E−08 293 CGPG5783 TPT 272 442 71.5 2.40E−18 294 CGPG5791 AA_permease 114 588 704.9 5.40E−209 295 CGPG5799 Aa_trans 206 601 453 3.60E−133 296 CGPG5856 Pkinase 79 353 159.5 8.00E−45 296 CGPG5856 Pkinase_Tyr 79 353 149.4 9.00E−42 297 CGPG5927 AAA 58 248 252.4 8.70E−73 297 CGPG5927 AAA 322 510 290.8 2.40E−84 298 CGPG5941 PfkB 42 336 86.2 9.00E−23 299 CGPG5957 CBM_20 86 178 23 5.70E−07 300 CGPG5967 DUF822 2 147 307.1 2.90E−89 301 CGPG6040 LEA_3 1 88 178.7 1.30E−50 302 CGPG607 PurA 28 275 44.4 5.80E−12 303 CGPG6178 DUF1336 53 267 427.9 1.30E−125 304 CGPG6185 UQ_con 7 148 197.9 2.10E−56 305 CGPG6306 APC8 1 161 401.5 1.10E−117 305 CGPG6306 TPR_1 339 372 34.7 2.90E−07 305 CGPG6306 TPR_2 339 372 23.8 0.00058 305 CGPG6306 TPR_2 373 406 23.7 0.00062 305 CGPG6306 TPR_1 373 406 34.1 4.40E−07 305 CGPG6306 TPR_2 407 440 22 0.0019 305 CGPG6306 TPR_1 407 440 24.3 0.00039 306 CGPG6318 MFS_1 31 496 35.3 1.90E−07 306 CGPG6318 PTR2 92 484 208.6 1.30E−59 307 CGPG6326 Kelch_1 34 79 45 2.40E−10 307 CGPG6326 Kelch_2 34 79 43.5 6.80E−10 307 CGPG6326 Kelch_1 152 198 26.7 7.70E−05 307 CGPG6326 Kelch_2 152 198 32.1 1.80E−06 307 CGPG6326 Kelch_2 203 249 20.2 0.0067 307 CGPG6326 Kelch_1 204 248 7.9 0.45 308 CGPG6370 Gp_dh_N 3 151 326 5.80E−95 308 CGPG6370 Gp_dh_C 156 313 362 8.80E−106 309 CGPG6429 ADH_N 25 135 159.4 8.40E−45 309 CGPG6429 ADH_zinc_N 166 305 105.1 1.90E−28 310 CGPG6440 PK 1 345 789.4 1.90E−234 310 CGPG6440 PK_C 357 471 167.5 3.10E−47 310 CGPG6440 PEP-utilizers 486 575 134 3.60E−37 311 CGPG6516 Aldedh 19 478 778.3 4.10E−231 312 CGPG6653 LRRNT_2 23 66 49.3 1.20E−11 312 CGPG6653 LRR_1 71 93 12.1 1.4 312 CGPG6653 LRR_1 95 117 10.5 2.8 312 CGPG6653 LRR_1 119 142 13 0.93 312 CGPG6653 LRR_1 144 166 19.5 0.011 312 CGPG6653 LRR_1 168 190 10.6 2.7 312 CGPG6653 LRR_1 192 214 8.8 5.7 312 CGPG6653 LRR_1 289 311 17.4 0.046 312 CGPG6653 LRR_1 313 335 10.8 2.4 312 CGPG6653 LRR_1 337 359 10.7 2.5 312 CGPG6653 LRR_1 361 384 12 1.5 312 CGPG6653 LRR_1 409 431 10.4 2.9 312 CGPG6653 LRR_1 457 479 11.9 1.5 312 CGPG6653 LRR_1 481 503 10.4 3 312 CGPG6653 LRR_1 505 527 10.5 2.8 312 CGPG6653 LRR_1 529 551 11.1 2.2 312 CGPG6653 LRR_1 553 575 9.3 4.7 312 CGPG6653 LRR_1 577 598 11.1 2.1 312 CGPG6653 Pkinase 695 966 134.8 2.20E−37 312 CGPG6653 Pkinase_Tyr 695 966 134.5 2.60E−37 313 CGPG6712 PGAM 91 277 154.6 2.30E−43 314 CGPG6737 PGAM 92 253 173 6.80E−49 315 CGPG6747 FBPase 106 429 448.6 7.20E−132 316 CGPG6796 Alpha-amylase 14 452 199.5 7.00E−57 318 CGPG6810 GH3 12 561 1261.5 0 320 CGPG6953 Ank 121 153 50.4 5.60E−12 321 CGPG7121 L51_S25_Cl-B8 20 93 108.9 1.40E−29 322 CGPG7163 Prenylcys_lyase 149 500 788 5.20E−234 324 CGPG7206 Aldo_ket_red 14 298 389.4 4.80E−114 325 CGPG7225 Subtilisin_N 48 125 84.3 3.30E−22 326 CGPG7267 DUF588 34 164 154.6 2.40E−43 327 CGPG7272 DUF1005 50 254 524.8 8.50E−155 328 CGPG7281 FA_hydroxylase 86 229 361.9 9.30E−106 329 CGPG7308 CoA_binding 1 100 −10.8 0.04 329 CGPG7308 NAD_GlySP_dh_N 4 147 −0.6 2.00E−06 329 CGPG7308 F420_oxidored 5 251 282.5 7.50E−82 330 CGPG7316 Anti-silence 1 155 419.9 3.20E−123 331 CGPG7371 Response_reg 29 157 94.8 2.30E−25 332 CGPG7457 PfkB 114 408 146.6 6.00E−41 335 CGPG7636 LSM 13 81 78.2 2.40E−20 338 CGPG7804 FAR1 62 279 364.9 1.20E−106 338 CGPG7804 SWIM 556 589 37.3 4.90E−08 339 CGPG7823 Rotamase 104 188 91.8 1.90E−24 339 CGPG7823 Rhodanese 203 298 44.1 4.40E−10 340 CGPG7828 DnaJ 12 81 66.1 1.00E−16 340 CGPG7828 zf-CSL 96 174 25.2 0.00021 343 CGPG7986 F-box 48 96 31.2 3.40E−06 343 CGPG7986 LRR_1 189 216 9 5.4 343 CGPG7986 LRR_1 428 451 8.2 7.7 343 CGPG7986 LRR_1 561 584 8.4 6.9 345 CGPG8015 zf-CCHC 18 35 24.2 7.90E−05 349 CGPG8083 Tryp_alpha_amyl 28 105 37.6 3.90E−08 350 CGPG8106 BURP 56 280 380 3.20E−111 353 CGPG8152 PAP_fibrillin 9 124 45.9 1.20E−10 354 CGPG8166 PBD 118 166 49.9 7.80E−12 355 CGPG8377 Oleosin 30 109 55 2.20E−13 356 CGPG8976 Ceramidase_alk 50 795 1545.5 0 357 CGPG8987 FH2 439 839 552.9 3.10E−163 358 CGPG9013 NAD_binding_1 234 350 138.6 1.50E−38 359 CGPG9080 EGF_CA 315 357 42.2 1.60E−09 359 CGPG9080 Pkinase 433 716 120.3 4.90E−33 359 CGPG9080 Pkinase_Tyr 433 704 113.1 7.40E−31 360 CGPG9081 DUF676 30 247 319 7.50E−93 361 CGPG9130 MMR_HSR1 266 369 71.9 1.80E−18 362 CGPG9133 PPR 125 159 13.2 0.19 362 CGPG9133 PPR 161 195 2.8 3.2 362 CGPG9133 PPR 196 230 22.7 0.0012 362 CGPG9133 PPR 232 266 42.1 1.70E−09 362 CGPG9133 PPR 267 301 29.4 1.20E−05 362 CGPG9133 PPR 302 336 49.4 1.10E−11 362 CGPG9133 PPR 337 371 32.9 1.00E−06 362 CGPG9133 PPR 372 407 7.9 0.8 362 CGPG9133 PPR 408 442 49.7 8.80E−12 362 CGPG9133 PPR 443 477 22.6 0.0013 362 CGPG9133 PPR 478 512 37.8 3.40E−08 362 CGPG9133 PPR 513 547 42.9 1.00E−09 362 CGPG9133 PPR 548 582 30.2 6.70E−06 362 CGPG9133 PPR 583 617 38.4 2.20E−08 363 CGPG9134 HD 91 232 46.2 1.00E−10 364 CGPG9137 RrnaAD 65 337 122.2 1.30E−33 365 CGPG9141 Pantoate_transf 40 306 402.9 4.40E−118 366 CGPG9145 Lung_7-TM_R 168 423 385.2 8.80E−113 367 CGPG9147 DNA_pol_E_B 178 389 249.9 5.00E−72 368 CGPG9148 p450 36 502 128.8 1.40E−35 369 CGPG9155 Pkinase 86 347 56.9 6.10E−14 369 CGPG9155 Pkinase_Tyr 86 351 73.7 2.90E−20 370 CGPG9163 Na_H_Exchanger 12 378 280.1 3.80E−81 370 CGPG9163 TrkA_N 416 531 118.8 1.50E−32 371 CGPG9170 Complex1_30 kDa 90 158 103.8 4.70E−28 371 CGPG9170 Complex1_49 kDa 298 537 2.8 6.10E−13 373 CGPG9183 HTH_11 1 56 72.4 1.30E−18 373 CGPG9183 BPL_LipA_LipB 84 182 94.5 2.90E−25 373 CGPG9183 BPL_C 275 322 43.3 7.80E−10 374 CGPG9186 DHBP_synthase 8 203 370.6 2.20E−108 374 CGPG9186 GTP_cyclohydro2 208 366 −2.3 3.80E−10 375 CGPG9205 NTP_transferase 4 288 421 1.50E−123 375 CGPG9205 MannoseP_isomer 299 465 350.8 2.00E−102 375 CGPG9205 Cupin_2 380 450 55.3 1.80E−13 376 CGPG9207 HTH_11 6 59 50.9 3.90E−12 376 CGPG9207 BPL_LipA_LipB 83 180 104.5 2.80E−28 376 CGPG9207 BPL_C 271 317 46.5 8.50E−11 377 CGPG9219 Complex1_30 kDa 107 175 99.8 7.60E−27 377 CGPG9219 Complex1_49 kDa 311 537 −11.6 5.90E−12 378 CGPG9220 GDC-P 3 443 700.9 8.30E−208 379 CGPG9230 Peptidase_S10 88 488 657.2 1.20E−194 381 CGPG9238 Tryp_alpha_amyl 36 114 56 1.20E−13 382 CGPG9259 Mit_rib_S27 14 93 135.3 1.50E−37 383 CGPG9271 NPH3 215 418 189.9 5.60E−54 384 CGPG9275 ETC_C1_NDUFA5 35 91 112.8 9.00E−31 386 CGPG9283 DUF1195 6 161 180.3 4.30E−51 387 CGPG9309 MAP65_ASE1 38 575 52.8 1.10E−12 389 CGPG9322 Pkinase 103 383 170 5.40E−48 389 CGPG9322 Pkinase_Tyr 103 383 158.7 1.30E−44 390 CGPG9335 Sugar_tr 7 726 210.6 3.30E−60 390 CGPG9335 MFS_1 11 685 107.7 3.00E−29 391 CGPG9341 RMMBL 531 573 35.9 1.20E−07 392 CGPG9344 TPR_2 531 564 23.9 0.00051 393 CGPG9345 UPF0261 5 432 483.7 2.00E−142 394 CGPG976 Glyco_transf_8 216 533 401.3 1.30E−117

TABLE 17 Pfam domain accession gathering name number cutoff domain description 14-3-3 PF00244.9 25 14-3-3 protein 2-Hacid_dh PF00389.19 11.2 D-isomer specific 2-hydroxyacid dehydrogenase, catalytic domain 2-Hacid_dh_C PF02826.7 −82.2 D-isomer specific 2-hydroxyacid dehydrogenase, NAD binding domain AAA PF00004.18 12.3 ATPase family associated with various cellular activities (AAA) AA_permease PF00324.10 −120.8 Amino acid permease ACT PF01842.13 0 ACT domain ADH_N PF08240.2 −14.5 Alcohol dehydrogenase GroES-like domain ADH_zinc_N PF00107.16 23.8 Zinc-binding dehydrogenase APC8 PF04049.3 −19.8 Anaphase promoting complex subunit 8/ Cdc23 Aa_trans PF01490.7 −128.4 Transmembrane amino acid transporter protein Actin PF00022.8 −30 Actin Aldedh PF00171.11 −209.3 Aldehyde dehydrogenase family Aldo_ket_red PF00248.10 −97 Aldo/keto reductase family Allene_ox_cyc PF06351.2 25 Allene oxide cyclase Alpha-amylase PF00128.12 −93 Alpha amylase, catalytic domain Aminotran_1_2 PF00155.10 −57.5 Aminotransferase class I and II Aminotran_3 PF00202.10 −207.6 Aminotransferase class-III Ank PF00023.18 0 Ankyrin repeat Anti-silence PF04729.4 25 Anti-silencing protein, ASF1-like ArfGap PF01412.8 −17 Putative GTPase activating protein for Arf Asp PF00026.13 −186.1 Eukaryotic aspartyl protease Auxin_inducible PF02519.4 −15 Auxin responsive protein BPL_C PF02237.6 16 Biotin protein ligase C terminal domain BPL_LipA_LipB PF03099.8 −0.2 Biotin/lipoate A/B protein ligase family BURP PF03181.5 −52 BURP domain B_lectin PF01453.14 28.2 D-mannose binding lectin BolA PF01722.7 23 BolA-like protein C2 PF00168.18 3.7 C2 domain CBM_20 PF00686.9 −3 Starch binding domain CMAS PF02353.10 −177.9 Cyclopropane-fatty-acyl-phospholipid synthase Ceramidase_alk PF04734.3 25 Neutral/alkaline non-lysosomal ceramidase CoA_binding PF02629.8 −12.8 CoA binding domain Complex1_30kDa PF00329.8 −3 Respiratory-chain NADH dehydrogenase, 30 Kd subunit Complex1_49kDa PF00346.8 −108 Respiratory-chain NADH dehydrogenase, 49 Kd subunit Cupin_2 PF07883.1 16.6 Cupin domain Cupin_3 PF05899.2 4.4 Protein of unknown function (DUF861) Cyclin_C PF02984.8 −13 Cyclin, C-terminal domain Cyclin_N PF00134.13 −14.7 Cyclin, N-terminal domain DHBP_synthase PF00926.10 −116 3,4-dihydroxy-2-butanone 4-phosphate synthase DNA_pol_E_B PF04042.5 −47.5 DNA polymerase alpha/epsilon subunit B DUF1005 PF06219.2 25 Protein of unknown function (DUF1005) DUF1191 PF06697.2 25 Protein of unknown function (DUF1191) DUF1195 PF06708.1 25 Protein of unknown function (DUF1195) DUF1336 PF07059.2 −78.2 Protein of unknown function (DUF1336) DUF26 PF01657.7 0 Domain of unknown function DUF26 DUF298 PF03556.6 25 Domain of unknown function (DUF298) DUF588 PF04535.2 25 Domain of unknown function (DUF588) DUF676 PF05057.4 −60.7 Putative serine esterase (DUF676) DUF822 PF05687.3 25 Plant protein of unknown function (DUF822) DUF868 PF05910.2 25 Plant protein of unknown function (DUF868) DnaJ PF00226.19 −8 DnaJ domain EGF_CA PF07645.4 24.5 Calcium binding EGF domain ESCRT-III PF03357.10 −35.4 ESCRT-III complex subunit ETC_C1_NDUFA5 PF04716.3 25 ETC complex I subunit conserved region F-box PF00646.21 13.6 F-box domain F420_oxidored PF03807.6 −34.5 NADP oxidoreductase coenzyme F420- dependent FAR1 PF03101.4 0 FAR1 family FA_hydroxylase PF04116.2 −64.1 Fatty acid hydroxylase FBPase PF00316.10 −170.3 Fructose-1-6-bisphosphatase FH2 PF02181.13 −98.3 Formin Homology 2 Domain GDC-P PF02347.5 −306.2 Glycine cleavage system P-protein GH3 PF03321.3 −336 GH3 auxin-responsive promoter GTP_cyclohydro2 PF00925.11 −49 GTP cyclohydrolase II Gln-synt_C PF00120.14 −124 Glutamine synthetase, catalytic domain Gln-synt_N PF03951.9 9 Glutamine synthetase, beta-Grasp domain Glyco_transf_8 PF01501.9 −43.2 Glycosyl transferase family 8 Glycolytic PF00274.9 −174.5 Fructose-bisphosphate aldolase class-I Gp_dh_C PF02800.9 −64.1 Glyceraldehyde 3-phosphate dehydrogenase, C-terminal domain Gp_dh_N PF00044.12 −74.2 Glyceraldehyde 3-phosphate dehydrogenase, NAD binding domain HD PF01966.11 18 HD domain HLH PF00010.15 8.2 Helix-loop-helix DNA-binding domain HTH_11 PF08279.1 11.3 HTH domain Histone PF00125.13 17.4 Core histone H2A/H2B/H3/H4 Kelch_1 PF01344.14 7.8 Kelch motif Kelch_2 PF07646.4 14 Kelch motif L51_S25_CI-B8 PF05047.5 25 Mitochondrial ribosomal protein L51/S25/ CI-B8 domain LEA_3 PF03242.3 25 Late embryogenesis abundant protein LMBR1 PF04791.5 −116.3 LMBR1-like membrane protein LRRNT_2 PF08263.2 18.6 Leucine rich repeat N-terminal domain LRR_1 PF00560.21 7.7 Leucine Rich Repeat LSM PF01423.12 13.7 LSM domain Lung_7-TM_R PF06814.3 25 Lung seven transmembrane receptor MAP65_ASE1 PF03999.2 −134.8 Microtubule associated protein (MAP65/ASE1 family) MFS_1 PF07690.5 23.5 Major Facilitator Superfamily MMR_HSR1 PF01926.11 31.2 GTPase of unknown function MannoseP_isomer PF01050.8 −70 Mannose-6-phosphate isomerase Metalloenzyme PF01676.7 −14.4 Metalloenzyme superfamily Metallophos PF00149.17 22 Calcineurin-like phosphoesterase Methyltransf_11 PF08241.1 17.1 Methyltransferase domain Methyltransf_12 PF08242.1 21.4 Methyltransferase domain Mit_rib_S27 PF08293.1 25 Mitochondrial ribosomal subunit S27 MtN3_slv PF03083.5 −0.8 MtN3/saliva family NAD_Gly3P_dh_N PF01210.12 −44 NAD-dependent glycerol-3-phosphate dehydrogenase N-terminus NAD_binding_1 PF00175.10 −3.9 Oxidoreductase NAD-binding domain NAF PF03822.4 4.5 NAF domain NDK PF00334.9 −59.9 Nucleoside diphosphate kinase NPH3 PF03000.4 25 NPH3 family NTP_transferase PF00483.12 −90.5 Nucleotidyl transferase Na_H_Exchanger PF00999.10 −67.9 Sodium/hydrogen exchanger family ORMDL PF04061.4 25 ORMDL family Oleosin PF01277.7 −27 Oleosin PAN_2 PF08276.2 −4.9 PAN-like domain PAP_fibrillin PF04755.2 25 PAP_fibrillin PBD PF00786.17 12.2 P21-Rho-binding domain PCI PF01399.15 25 PCI domain PEP-utilizers PF00391.12 10 PEP-utilising enzyme, mobile domain PGAM PF00300.12 −3 Phosphoglycerate mutase family PK PF00224.10 −244 Pyruvate kinase, barrel domain PK_C PF02887.5 −44 Pyruvate kinase, alpha/beta domain PPR PF01535.11 0 PPR repeat PTR2 PF00854.12 −50 POT family Pantoate_transf PF02548.5 −93 Ketopantoate hydroxymethyltransferase Peptidase_S10 PF00450.11 −198 Serine carboxypeptidase PfkB PF00294.13 −67.8 pfkB family carbohydrate kinase Phosphorylase PF00343.9 −601.1 Carbohydrate phosphorylase Pkinase PF00069.14 −70.8 Protein kinase domain Pkinase_Tyr PF07714.5 65 Protein tyrosine kinase Prenylcys_lyase PF07156.3 −164.1 Prenylcysteine lyase PurA PF04845.3 25 PurA ssDNA and RNA-binding protein RMMBL PF07521.1 18.5 RNA-metabolising metallo-beta-lactamase Response_reg PF00072.12 4 Response regulator receiver domain Rhodanese PF00581.9 25 Rhodanese-like domain Rib_5-P_isom_A PF06026.4 25 Ribose 5-phosphate isomerase A (phosphoriboisomerase A) Ribonuclease_T2 PF00445.8 −53 Ribonuclease T2 family Ribosomal_S8e PF01201.11 25 Ribosomal protein S8e Rotamase PF00639.10 4 PPIC-type PPIASE domain RrnaAD PF00398.9 −73.3 Ribosomal RNA adenine dimethylase SAM_1 PF00536.18 11.3 SAM domain (Sterile alpha motif) SMC_N PF02463.8 −95.8 RecF/RecN/SMC N terminal domain SNF5 PF04855.3 25 SNF5/SMARCB1/INI1 SWIB PF02201.9 −7 SWIB/MDM2 domain SWIM PF04434.7 10 SWIM zinc finger S_locus_glycop PF00954.11 −12.7 S-locus glycoprotein family Sad1_UNC PF07738.2 −20.4 Sad1/UNC-like C-terminal Sterol_MT_C PF08498.1 30.5 Sterol methyltransferase C-terminal Subtilisin_N PF05922.6 26.1 Subtilisin N-terminal Region Sugar_tr PF00083.13 −85 Sugar (and other) transporter TFIIA PF03153.4 25 Transcription factor IIA, alpha/beta subunit TFIIS_C PF01096.9 15 Transcription factor S-II (TFIIS) TFIIS_M PF07500.3 7.4 Transcription factor S-II (TFIIS), central domain TIM PF00121.8 −97 Triosephosphate isomerase TPR_1 PF00515.16 7.7 Tetratricopeptide repeat TPR_2 PF07719.5 20.1 Tetratricopeptide repeat TPT PF03151.7 −15.3 Triose-phosphate Transporter family Thioredoxin PF00085.9 −25.7 Thioredoxin Tim17 PF02466.8 2.7 Tim17/Tim22/Tim23 family TrkA_N PF02254.7 4.7 TrkA-N domain Tryp_alpha_amyl PF00234.10 −4 Protease inhibitor/seed storage/LTP family UPF0261 PF06792.1 25 Uncharacterised protein family (UPF0261) UQ_con PF00179.16 −30 Ubiquitin-conjugating enzyme Usp PF00582.16 36.1 Universal stress protein family WD40 PF00400.20 21.5 WD domain, G-beta repeat adh_short PF00106.14 −17 short chain dehydrogenase eIF-4B PF06273.1 −205.7 Plant specific eukaryotic initiation factor 4B iPGM_N PF06415.3 −263.4 BPG-independent PGAM N-terminus (iPGM_N) p450 PF00067.11 −105 Cytochrome P450 zf-AN1 PF01428.6 0 AN1-like Zinc finger zf-C3HC4 PF00097.13 16.9 Zinc finger, C3HC4 type (RING finger) zf-CCCH PF00642.14 0 Zinc finger C-x8-C-x5-C-x3-H type (and similar) zf-CCHC PF00098.12 17.9 Zinc knuckle zf-CSL PF05207.3 2.9 CSL zinc finger

Example 5 Plasmid Construction for Transferring Recombinant DNA

This example illustrates the construction of plasmids for transferring recombinant DNA into the nucleus of a plant cell which can be regenerated into a transgenic crop plant of this invention. Primers for PCR amplification of protein coding nucleotides of recombinant DNA are designed at or near the start and stop codons of the coding sequence, in order to eliminate most of the 5′ and 3′ untranslated regions. DNA of interest, i.e. each DNA identified in Table 1 and the DNA for the identified homologous genes, are cloned and amplified by PCR prior to insertion into the insertion site the base vector.

A. Corn Transformation Vector

Elements of an exemplary common expression vector, pMON93093 are illustrated in Table 18. The exemplary base vector which is especially useful for corn transformation is illustrated in FIG. 2 and assembled using technology known in the art. The DNA of interest are inserted in a expression vector at the insertion site between the intron1 of rice act 1 gene and the termination sequence of PinII gene.

TABLE 18 pMON93093 Coordinates of SEQ ID NO: function name annotation 19940 Agro B-AGRtu.right border Agro right border 11364-11720 transforamtion sequence, essential for transfer of T-DNA. Gene of E-Os.Act1 upstream promoter  19-775 interest region of the rice actin expression 1 gene cassette E-CaMV.35S.2xA1-B3 duplicated35S A1-B3  788-1120 domain without TATA box P-Os.Act1 promoter region of the 1125-1204 rice actin 1 gene L-Ta.Lhcb1 5′ untranslated leader 1210-1270 of wheat major chlorophyll a/b binding protein I-Os.Act1 first intron and flanking 1287-1766 UTR exon sequences from the rice actin 1 gene T-St.Pis4 3′ non-translated region 1838-2780 of the potato proteinase inhibitor II gene which functions to direct polyadenylation of the mRNA Plant P-Os.Act1 Promoter from the rice 2830-3670 selectable actin 1 gene marker L-Os.Act1 first exon of the rice 3671-3750 expression actin 1 gene cassette I-Os.Act1 first intron and flanking 3751-4228 UTR exon sequences from the rice actin 1 gene TS-At.ShkG-CTP2 Transit peptide region 4238-4465 of Arabidopsis EPSPS CR-AGRtu.aroA- Synthetic CP4 coding 4466-5833 CP4.nat region with dicot preferred codon usage. T-AGRtu.nos A 3′ non-translated 5849-6101 region of the nopaline synthase gene of Agrobacterium tumefaciens Ti plasmid which functions to direct polyadenylation of the mRNA. Agro B-AGRtu.left border Agro left border 6168-6609 transformation sequence, essential for transfer of T-DNA. Maintenance OR-Ec.oriV-RK2 The vegetative origin of 6696-7092 in E. coli replication from plasmid RK2. CR-Ec.rop Coding region for 8601-8792 represser of primer from the ColE1 plasmid. Expression of this gene product interferes with primer binding at the origin of replication, keeping plasmid copy number low. OR-Ec.ori-ColE1 The minimal origin of 9220-9808 replication from the E. coli plasmid ColE1. P-Ec.aadA-SPC/STR remoter for Tn7 10339-10380 adenylyltransferase (AAD(3″)) CR-Ec.aadA-SPC/STR Coding region for Tn7 10381-11169 adenylyltransferase (AAD(3″)) conferring spectinomycin and streptomycin resistance. T-Ec.aadA-SPC/STR 3′ UTR from the Tn7 11170-11227 adenylyltransferase (AAD(3″))gene of E. coli.

B. Soybean Transformation Vector

Plasmids for use in transformation of soybean are also prepared. Elements of an exemplary common expression vector plasmid pMON82053 are shown in Table 19 below. This exemplary soybean or canola transformation base vector illustrated in FIG. 3 is assembled using the technology known in the art. Recobminant DNA of interest, i.e. each DNA identified in Table 1 and the DNA for the identified homologous genes, is cloned and amplified by PCR prior to insertion into the insertion site the base vector at the insertion site between the enhanced 35S CaMV promoter and the termination sequence of cotton E6 gene.

TABLE 19 pMON82053 Coordinates of SEQ ID NO: function name annotation 19941 Agro B-AGRtu.left Agro left border sequence, essential 6144-6585 transforamtion border for transfer of T-DNA. Plant P-At.Act7 Promoter from the arabidopsis actin 6624-7861 selectable 7 gene marker L-At.Act7 5′UTR of Arabidopsis Act7 gene expression I-At.Act7 Intron from the Arabidopsis actin7 cassette gene TS-At.ShkG- Transit peptide region of Arabidopsis 7864-8091 CTP2 EPSPS CR-AGRtu.aroA- Synthetic CP4 coding region with 8092-9459 CP4.nno_At dicot preferred codon usage. T-AGRtu.nos A 3′ non-translated region of the 9466-9718 nopaline synthase gene of Agrobacterium tumefaciens Ti plasmid which functions to direct polyadenylation of the mRNA. Gene of P-CaMV.35S-enh Promoter for 35S RNA from CaMV  1-613 interest containing a duplication of the −90 expression to −350 region. cassette T-Gb.E6-3b 3′ untranslated region from the fiber  688-1002 protein E6 gene of sea-island cotton; Agro B-AGRtu.right Agro right border sequence, essential 1033-1389 transformation border for transfer of T-DNA. Maintenance OR-Ec.oriV-RK2 The vegetative origin of replication 5661-6057 in E. coli from plasmid RK2. CR-Ec.rop Coding region for represser of primer 3961-4152 from the ColE1 plasmid. Expression of this gene product interferes with primer binding at the origin of replication, keeping plasmid copy number low. OR-Ec.ori-ColE1 The minimal origin of replication 2945-3533 from the E. coli plasmid ColE1. P-Ec.aadA- romoter for Tn7 adenylyltransferase 2373-2414 SPC/STR (AAD(3″)) CR-Ec.aadA- Coding region for Tn7 1584-2372 SPC/STR adenylyltransferase (AAD(3″)) conferring spectinomycin and streptomycin resistance. T-Ec.aadA- 3′ UTR from the Tn7 1526-1583 SPC/STR adenylyltransferase (AAD(3″)) gene of E. coli.

C. Cotton Transformation Vector

Plasmids for use in transformation of cotton are also prepared. Elements of an exemplary common expression vector plasmid pMON99053 are shown in Table 20 below and FIG. 4. Primers for PCR amplification of protein coding nucleotides of recombinant DNA are designed at or near the start and stop codons of the coding sequence, in order to eliminate most of the 5′ and 3′ untranslated regions. Each recombinant DNA coding for a protein identified in Table 1 is amplified by PCR prior to insertion into the insertion site within the gene of interest expression cassette of one of the base.

TABLE 20 Coordinates of SEQ ID function name annotation NO: 19942 Agro B-AGRtu.right border Agro right border sequence, 11364-11720 transforamtion essential for transfer of T-DNA. Gene of interest Exp-CaMV.35S- Enhanced version of the 35S 7794-8497 expression enh + ph.DnaK RNA promoter from CaMV cassette plus the petunia hsp70 5′ untranslated region T-Ps.RbcS2-E9 The 3′ non-translated region  67-699 of the pea RbcS2 gene which functions to direct polyadenylation of the mRNA. Plant selectable Exp-CaMV.35S Promoter and 5′ untranslated  730-1053 marker region of the 35S RNA from expression CaMV cassette CR-Ec.nptII-Tn5 Neomycin 1087-1881 Phosphotransferase II gene that confers resistance to neomycin and kanamycin T-AGRtu.nos A 3′ non-translated region of 1913-2165 the nopaline synthase gene of Agrobacterium tumefaciens Ti plasmid which functions to direct polyadenylation of the mRNA. Agro B-AGRtu.left border Agro left border sequence, 2211-2652 transformation essential for transfer of T-DNA. Maintenance in OR-Ec.oriV-RK2 The vegetative origin of 2739-3135 E. coli replication from plasmid RK2. CR-Ec.rop Coding region for represser 4644-4835 of primer from the ColE1 plasmid. Expression of this gene product interferes with primer binding at the origin of replication, keeping plasmid copy number low. OR-Ec.ori-ColE1 The minimal origin of 5263-5851 replication from the E. coli plasmid ColE1. P-Ec.aadA-SPC/STR romoter for Tn7 6382-6423 adenylyltransferase (AAD(3″)) CR-Ec.aadA-SPC/STR Coding region for Tn7 6424-7212 adenylyltransferase (AAD(3″)) conferring spectinomycin and streptomycin resistance. T-Ec.aadA-SPC/STR 3′ UTR from the Tn7 7213-7270 adenylyltransferase (AAD(3″)) gene of E. coli.

Example 6 Corn Plant Transformation

This example illustrates the production and identification of transgenic corn cells in seed of transgenic corn plants having an enhanced agronomic trait, i.e. enhanced nitrogen use efficiency, increased yield, enhanced water use efficiency, enhanced tolerance to cold and/or improved seed compositions as compared to control plants. Transgenic corn cells are prepared with recombinant DNA expressing each of the protein encoding DNAs listed in Table 1 by Agrobacterium-mediated transformation using the corn transformation vectors pMON93093 as disclosed in Example 6. Corn transformation is effected using methods disclosed in U.S. Patent Application Publication 2004/0344075 A1 where corn embryos are inoculated and co-cultured with the Agrobacterium tumefaciens strain ABI and the corn transformation vector. To regenerate transgenic corn plants the transgenic callus resulting from transformation is placed on media to initiate shoot development in plantlets which are transferred to potting soil for initial growth in a growth chamber followed by a mist bench before transplanting to pots where plants are grown to maturity. The plants are self fertilized and seed is harvested for screening as seed, seedlings or progeny R2 plants or hybrids, e.g., for yield trials in the screens indicated above.

Many transgenic events which survive to fertile transgenic plants that produce seeds and progeny plants do not exhibit an enhanced agronomic trait. The transgenic plants and seeds having the transgenic cells of this invention which have recombinant DNA imparting the enhanced agronomic traits are identified by screening for nitrogen use efficiency, yield, water use efficiency, cold tolerance and improved seed composition.

Example 7 Soybean Plant Transformation

This example illustrates the production and identification of transgenic soybean cells in seed of transgenic soybean plants having an enhanced agronomic trait, i.e. enhanced nitrogen use efficiency, increased yield, enhanced water use efficiency, enhanced tolerance to cold and/or improved seed compositions as compared to control plants. Transgenic soybean cells are prepared with recombinant DNA expressing each of the protein encoding DNAs listed in Table 1 by Agrobacterium-mediated transformation using the soybean transformation vectors pMON82053 disclosed in Example 7. Soybean transformation is effected using methods disclosed in U.S. Pat. No. 6,384,301 where soybean meristem explants are wounded then inoculated and co-cultured with the soybean transformation vector, then transferred to selection media for 6-8 weeks to allow selection and growth of transgenic shoots.

The transformation is repeated for each of the protein encoding DNAs identified in Table 1.

Transgenic shoots producing roots are transferred to the greenhouse and potted in soil. Many transgenic events which survive to fertile transgenic plants that produce seeds and progeny plants do not exhibit an enhanced agronomic trait. The transgenic plants and seeds having the transgenic cells of this invention which have recombinant DNA imparting the enhanced agronomic traits are identified by screening for nitrogen use efficiency, yield, water use efficiency, cold tolerance and improved seed composition.

Example 8 Canola Transformation

This example illustrates plant transformation useful in producing the transgenic canola plants of this invention and the production and identification of transgenic seed for transgenic canola having enhanced water use efficiency, enhanced cold tolerance, increased yield, enhanced nitrogen use efficiency, enhanced seed protein and enhanced seed oil.

Tissues from in vitro grown canola seedlings are prepared and inoculated with overnight-grown Agrobacterium cells containing plasmid DNA with the gene of interest cassette and a plant selectable marker cassette. Following co-cultivation with Agrobacterium, the infected tissues are allowed to grow on selection to promote growth of transgenic shoots, followed by growth of roots from the transgenic shoots. The selected plantlets are then transferred to the greenhouse and potted in soil. Molecular characterization are performed to confirm the presence of the gene of interest, and its expression in transgenic plants and progenies. Progeny transgenic plants are selected from a population of transgenic canola events under specified growing conditions and are compared with control canola plants. Control canola plants are substantially the same canola genotype but without the recombinant DNA, for example, either a parental canola plant of the same genotype that is not transformed with the identical recombinant DNA or a negative isoline of the transformed plant

Transgenic canola plant cells are transformed with recombinant DNA from each of the genes identified in Table 1. Transgenic progeny plants and seed of the transformed plant cells are screened for enhanced water use efficiency, enhanced cold tolerance, increased yield, enhanced nitrogen use efficiency, enhanced seed protein and enhanced seed oil as reported in Example 9.

Example 9 Selection of Transgenic Plants with Enhanced Agronomic Trait(s)

This example illustrates identification of nuclei of the invention by screening derived plants and seeds for an enhanced trait identified below.

Many transgenic events which survive to fertile transgenic plants that produce seeds and progeny plants will not exhibit an enhanced agronomic trait. Populations of transgenic seed and plants prepared in Examples 6 and 7 are screened to identify those transgenic events providing transgenic plant cells with a nucleus having recombinant DNA imparting an enhanced trait. Each population is screened for enhanced nitrogen use efficiency, increased yield, enhanced water use efficiency, enhanced tolerance to cold and heat, increased level of oil and protein in seed using assays described below. Plant cell nuclei having recombinant DNA with each of the genes identified in Table 1 and the identified homologs are identified in plants and seeds with at least one of the enhanced traits.

A. Selection for Enhanced Nitrogen Use Efficiency

Transgenic corn plants with nuclei of the invention are planted in fields with three levels of nitrogen (N) fertilizer being applied, i.e. low level (0 pounds per acre N), medium level (80 pounds per acre N) and high level (180 pounds per acre N). Liquid 28% or 32% UAN (Urea, Ammonium Nitrogen) are used as the N source and apply by broadcast boom and incorporate with a field cultivator with rear rolling basket in the same direction as intended crop rows. Although there is no N applied in the low level treatment, the soil should still be disturbed in the same fashion as the treated area. Transgenic plants and control plants can be grouped by genotype and construct with controls arranged randomly within genotype blocks. For improved statistical analysis each type of transgenic plant can be tested by 3 replications and across 4 locations. Nitrogen levels in the fields are analyzed before planting by collecting sample soil cores from 0-24″ and 24 to 48″ soil layer. Soil samples are analyzed for nitrate-nitrogen, phosphorus (P), potassium (K), organic matter and pH to provide baseline values. P, K and micronutrients are applied based upon soil test recommendations.

Transgenic corn plants prepared in Example 6 and which exhibit a 2 to 5% yield increase as compared to control plants when grown in the high nitrogen field are selected as having nuclei of the invention. Transgenic corn plants which have at least the same or higher yield as compared to control plants when grown in the medium nitrogen field are selected as having nuclei of the invention. Transgenic corn plants having a nucleus with DNA identified in Table 3 as imparting nitrogen use efficiency (LN) and homologous DNA are selected from a nitrogen use efficiency screen as having a nucleus of this invention.

B. Selection for Increased Yield

Many transgenic plants of this invention exhibit improved yield as compared to a control plant. Improved yield can result from enhanced seed sink potential, i.e. the number and size of endosperm cells or kernels and/or enhanced sink strength, i.e. the rate of starch biosynthesis. Sink potential can be established very early during kernel development, as endosperm cell number and size are determined within the first few days after pollination.

Much of the increase in corn yield of the past several decades has resulted from an increase in planting density. During that period, corn yield has been increasing at a rate of 2.1 bushels/acre/year, but the planting density has increased at a rate of 250 plants/acre/year. A characteristic of modern hybrid corn is the ability of these varieties to be planted at high density. Many studies have shown that a higher than current planting density should result in more biomass production, but current germplasm does not perform. well at these higher densities. One approach to increasing yield is to increase harvest index (HI), the proportion of biomass that is allocated to the kernel compared to total biomass, in high density plantings.

Effective yield selection of enhanced yielding transgenic corn events uses hybrid progeny of the transgenic event over multiple locations with plants grown under optimal production management practices, and maximum pest control. A useful target for improved yield is a 5% to 10% increase in yield as compared to yield produced by plants grown from seed for a control plant. Selection methods may be applied in multiple and diverse geographic locations, for example up to 16 or more locations, over one or more planting seasons, for example at least two planting seasons to statistically distinguish yield improvement from natural environmental effects. It is to plant multiple transgenic plants, positive and negative control plants, and pollinator plants in standard plots, for example 2 row plots, 20 feet long by 5 feet wide with 30 inches distance between rows and a 3 foot alley between ranges. Transgenic events can be grouped by recombinant DNA constructs with groups randomly placed in the field. A pollinator plot of a high quality corn line is planted for every two plots to allow open pollination when using male sterile transgenic events. A useful planting density is about 30,000 plants/acre. High planting density is greater than 30,000 plants/acre, preferably about 40,000 plants/acre, more preferably about 42,000 plants/acre, most preferably about 45,000 plants/acre. Each of the transgenic corn plants and soybean plants with a nucleus of the invention prepared in Examples 6 and 7 are screened for yield enhancement. At least one event from each of the corn and soybean plants is selected as having at least between 3 and 5% increase in yield as compared to a control plant as having a nucleus of this invention.

C. Selection for Enhanced Water Use Efficiency (WUE)

The following is a high-throughput method for screening for water use efficiency in a greenhouse to identify the transgenic corn plants with a nucleus of this invention. This selection process imposes 3 drought/re-water cycles on plants over a total period of 15 days after an initial stress free growth period of 11 days. Each cycle consists of 5 days, with no water being applied for the first four days and a water quenching on the 5th day of the cycle. The primary phenotypes analyzed by the selection method are the changes in plant growth rate as determined by height and biomass during a vegetative drought treatment. The hydration status of the shoot tissues following the drought is also measured. The plant height are measured at three time points. The first is taken just prior to the onset drought when the plant is 11 days old, which is the shoot initial height (SIR). The plant height is also measured halfway throughout the drought/re-water regimen, on day 18 after planting, to give rise to the shoot mid-drought height (SMH). Upon the completion of the final drought cycle on day 26 after planting, the shoot portion of the plant is harvested and measured for a final height, which is the shoot wilt height (SWH) and also measured for shoot wilted biomass (SWM). The shoot is placed in water at 40 degree Celsius in the dark. Three days later, the shoot is weighted to give rise to the shoot turgid weight (STM). After drying in an oven for four days, the shoots are weighted for shoot dry biomass (SDM). The shoot average height (SAH) is the mean plant height across the 3 height measurements. The procedure described above may be adjusted for +/−˜one day for each step given the situation.

To correct for slight differences between plants, a size corrected growth value is derived from SIH and SWH. This is the Relative Growth Rate (RGR). Relative Growth Rate (RGR) is calculated for each shoot using the formula [RGR %=(SWH−SIH)/((SWH+SIH)/2)×100]. Relative water content (RWC) is a measurement of how much (%) of the plant was water at harvest. Water Content (RWC) is calculated for each shoot using the formula [RWC %=(SWM−SDM)/(STM−SDM)×100]. Fully watered corn plants of this age run around 98% RWC.

Transgenic corn plants and soybean plants prepared in Examples 6 and 7 are screened for water use efficiency. Transgenic plants having at least a 1% increase in RGR and RWC as compared to control plants are identified as having enhanced water used efficiency and are selected as having a nucleus of this invention. Transgenic corn and soybean plants having in their nucleus DNA identified in Table 3 as imparting drought tolerance improvement (DS) and homologous DNA are identified as showing increased water use efficiency as compared to control plants and are selected as having a nucleus of this invention.

D. Selection for Growth Under Cold Stress

Cold germination assay—Three sets of seeds are used for the assay. The first set consists of positive transgenic events (F1 hybrid) where the genes of the present invention are expressed in the seed. The second seed set is nontransgenic, wild-type negative control made from the same genotype as the transgenic events. The third set consisted of two cold tolerant and one cold sensitive commercial check lines of corn. All seeds are treated with a fungicide “Captan” (MAESTRO® 80DF Fungicide, Arvesta Corporation, San Francisco, Calif., USA). 0.43 mL Captan is applied per 45 g of corn seeds by mixing it well and drying the fungicide prior to the experiment.

Corn kernels are placed embryo side down on blotter paper within an individual cell (8.9×8.9 cm) of a germination tray (54×36 cm). Ten seeds from an event are placed into one cell of the germination tray. Each tray can hold 21 transgenic events and 3 replicates of wildtype (LH244SDms+LH59), which is randomized in a complete block design. For every event there are five replications (five trays). The trays are placed at 9.7 C for 24 days (no light) in a Convrion® growth chamber (Conviron Model PGV36, Controlled Environments, Winnipeg, Canada). Two hundred and fifty milliliters of deionized water are added to each germination tray. Germination counts are taken 10th, 11th, 12th, 13th, 14th, 17th, 19th, 21st, and 24th day after start date of the experiment. Seeds are considered germinated if the emerged radicle size is 1 cm. From the germination counts germination index is calculated.

The germination index is calculated as per:

Germination index=(Σ([T+1−n _(i) ]*[P _(i) −P _(i−1)]))/T

where T is the total number of days for which the germination assay is performed. The number of days after planting is defined by n. “i” indicated the number of times the germination had been counted, including the current day. P is the percentage of seeds germinated during any given rating. Statistical differences are calculated between transgenic events and wild type control. After statistical analysis, the events that show a statistical significance at the p level of less than 0.1 relative to wild-type controls will advance to a secondary cold selection. The secondary cold screen is conducted in the same manner of the primary selection only increasing the number of repetitions to ten. Statistical analysis of the data from the secondary selection is conducted to identify the events that show a statistical significance at the p level of less than 0.05 relative to wild-type controls.

Transgenic corn plants and soybean plants prepared in Examples 6 and 7 are screened for water use efficiency. Transgenic plants having at least a 5% increase in germination index as compared to control plants are identified as having enhanced cold stress tolerance and are selected as having a nucleus of this invention. Transgenic corn and soybean plants having in their nucleus DNA identified in Table 3 as imparting cold tolerance improvement (CK or CS) and homologous DNA are identified as showing increased cold stress tolerance as compared to control plants and are selected as having a nucleus of this invention.

E. Screens for Transgenic Plant Seeds with Increased Protein and/or Oil Levels

The following is a high-throughput selection method for identifying plant seeds with improvement in seed composition using the Infratec® 1200 series Grain Analyzer, which is a near-infrared transmittance spectrometer used to determine the composition of a bulk seed sample. Near infrared analysis is a non-destructive, high-throughput method that can analyze multiple traits in a single sample scan. An NIR calibration for the analytes of interest is used to predict the values of an unknown sample. The NIR spectrum is obtained for the sample and compared to the calibration using a complex chemometric software package that provides a predicted values as well as information on how well the sample fits in the calibration.

Infratec® Model 1221, 1225, or 1227 analyzer with transport module by Foss North America is used with cuvette, item #1000-4033, Foss North America or for small samples with small cell cuvette, Foss standard cuvette modified by Leon Girard Co. Corn and soy check samples of varying composition maintained in check cell cuvettes are supplied by Leon Girard Co. NIT collection software is provided by Maximum Consulting Inc. Calculations are performed automatically by the software. Seed samples are received in packets or containers with barcode labels from the customer. The seed is poured into the cuvettes and analyzed as received.

TABLE 21 Typical sample(s): Whole grain corn and soybean seeds Analytical time to run method: Less than 0.75 min per sample Total elapsed time per run: 1.5 minute per sample Typical and minimum sample size: Corn typical: 50 cc; minimum 30 cc Soybean typical: 50 cc; minimum 5 cc Typical analytical range: Determined in part by the specific calibration. Corn - moisture 5-15%, oil 5-20%, protein 5-30%, starch 50-75%, and density 1.0-1.3%. Soybean - moisture 5-15%, oil 15-25%, and protein 35-50%. Transgenic corn plants and soybean plants prepared in Examples 6 and 7 are screened for increased protein and oil in seed. Transgenic inbred corn and soybean plants having an increase of at least 1 percentage point in the total percent seed protein or at least 0.3 percentage point in total seed oil and transgenic hybrid corn plants having an increase of at least 0.4 percentage point in the total percent seed protein as compared to control plants are identified as having enhanced seed protein or enhanced seed oil and are selected as having a nucleus of this invention.

Example 10 Cotton Transgenic Plants with Enhanced Agronomic Traits

Cotton transformation is performed as generally described in WO0036911 and in U.S. Pat. No. 5,846,797. Transgenic cotton plants containing each of the recombinant DNA having a sequence of SEQ ID NO: 1 through SEQ ID NO: 197 are obtained by transforming with recombinant DNA from each of the genes identified in Table 1. Progeny transgenic plants are selected from a population of transgenic cotton events under specified growing conditions and are compared with control cotton plants. Control cotton plants are substantially the same cotton genotype but without the recombinant DNA, for example, either a parental cotton plant of the same genotype that was not transformed with the identical recombinant DNA or a negative isoline of the transformed plant. Additionally, a commercial cotton cultivar adapted to the geographical region and cultivation conditions, i.e. cotton variety ST474, cotton variety FM 958, and cotton variety Siokra L-23, are used to compare the relative performance of the transgenic cotton plants containing the recombinant DNA. The specified culture conditions are growing a first set of transgenic and control plants under “wet” conditions, i.e. irrigated in the range of 85 to 100 percent of evapotranspiration to provide leaf water potential of −14 to −18 bars, and growing a second set of transgenic and control plants under “dry” conditions, i.e. irrigated in the range of 40 to 60 percent of evapotranspiration to provide a leaf water potential of −21 to −25 bars. Pest control, such as weed and insect control is applied equally to both wet and dry treatments as needed. Data gathered during the trial includes weather records throughout the growing season including detailed records of rainfall; soil characterization information; any herbicide or insecticide applications; any gross agronomic differences observed such as leaf morphology, branching habit, leaf color, time to flowering, and fruiting pattern; plant height at various points during the trial; stand density; node and fruit number including node above white flower and node above crack boll measurements; and visual wilt scoring. Cotton boll samples are taken and analyzed for lint fraction and fiber quality. The cotton is harvested at the normal harvest timeframe for the trial area. Enhanced water use efficiency is indicated by increased yield, improved relative water content, enhanced leaf water potential, increased biomass, enhanced leaf extension rates, and improved fiber parameters.

The transgenic cotton plants of this invention are identified from among the transgenic cotton plants by agronomic trait screening as having increased yield and enhanced water use efficiency.

Example 11 Monocot and Dicot Plant Transformation for the Suppression of Endogeneous Protein

This example illustrates monocot and dicot plant transformation to produce nuclei of this invention in cells of a transgenic plant by transformation where the recombinant DNA suppresses the expression of an endogenous protein identified by Pfam, SNF5, LMBR1, TFIIS_M, TFIIS_C, or Glyco_transf 8. Corn callus and soybean tissue are transformed as describe in Examples 6 and 7 using recombinant DNA in the nucleus with DNA that transcribes to RNA that forms double-stranded RNA targeted to an endogenous gene with DNA encoding the protein. The genes for which the double-stranded RNAs are targeted are the native gene in corn and soybean that are homolog of the genes encoding the protein with an amino acid sequence of SEQ ID NO:200, 201, 205, 207, 211, and 394.

Populations of transgenic corn plants and soybean plants prepared in Examples 6 and 7 with DNA for suppressing a gene identified in Table 3 as providing an enhanced trait by gene suppression are screened to identify an event from those plants with a nucleus of the invention by selecting the trait identified in this specification. 

1. A plant cell nucleus with stably integrated, recombinant DNA comprising a promoter that is functional in plant cells and that is operably linked to DNA from a plant, bacteria or yeast that encodes a protein having at least one domain of amino acids in a sequence that exceeds the Pfam gathering cutoff for amino acid sequence alignment with a protein domain family identified by a Pfam name selected from the group of Pfam names consisting of L51_S25_CI-B8, iPGM_N, WD40, BPL_LipA_LipB, DUF676, AAA, S_locus_glycop, ArfGap, Rotamase, Metallophos, CMAS, Sugar_tr, LMBR1, RrnaAD, NAF, BolA, Pkinase, C2, FA_hydroxylase, p450, Complex1_(—)30 kDa, Histone, DUF822, PEP-utilizers, PCI, ETC_C1_NDUFA5, 2-Hacid_dh, Tryp_alpha_amyl, PK_C, MAP65_ASE1, FBPase, SWIB, Ank, Ribosomal_S8e, 2-Hacid_dh_C, SMC_N, GTP_cyclohydro-2, PfkB, ORMDL, ADH_zinc_N, SWIM, TrkA_N, HLH, GH3, SNF5, Ceramidase_alk, Ribonuclease_T2, Complex1_(—)49 kDa, Gp_dh_C, Aldo_ket_red, zf-AN1, TFIIS_C, MFS_(—)1, Thioredoxin, DUF1005, LEA_(—)3, Sterol_MT_C, Gp_dh_N, TFIIS_M, PAN_(—)2, BPL_C, DUF26, Aa_trans, ACT, ADH_N, NAD_binding_(—)1, Auxin_inducible, B_lectin, Anti-silence, Response_reg, 14-3-3, LRRNT_(—)2, GDC-P, zf-CCHC, NPH3, TPR_(—)1, TFIIA, DHBP_synthase, UQ_con, TPR_(—)2, TPT, F-box, adh_short, Cyclin_C, Na_H_Exchanger, AA_permease, MtN3_slv, TIM, NDK, Pantoate_transf, Allene_ox_cyc, Cyclin_N, Methyltransf_(—)11, CBM_(—)20, Methyltransf_(—)12, Rhodanese, Glycolytic, Actin, Usp, eIF-4B, Glyco_transf 8, BURP, Alpha-amylase, F420_oxidored, EGF_CA, Kelch_(—)1, PGAM, Aminotran_(—)1_(—)2, Kelch_(—)2, UPF0261, CoA_binding, DUF868, Peptidase_S10, Lung_(—)7-TM_R, Oleosin, Sad1_UNC, Gln-synt_C, LSM, NTP_transferase, Metalloenzyme, Prenylcys_lyase, Subtilisin_N, SAM_(—)1, DUF298, ESCRT-III, DNA_pol_E_B, Aminotran_(—)3, NAD_Gly3P_dh_N, Gln-synt_N, MMR—HSR1, DUF588, zf-CCCH, DnaJ, Pkinase_Tyr, Cupin_(—)2, LRR_(—)1, Cupin_(—)3, zf-CSL, FAR1, HD, FH2, APC8, PTR₂, MannoseP_isomer, Rib_(—)5-P_isom_A, DUF1336, Phosphorylase, DUF1191, Asp, Mit_rib_(—)527, PAP_fibrillin, DUF1195, Aldedh, zf-C3HC4, PPR, PK, PurA, RMMBL, HTH_(—)11, Tim17, and PBD wherein said Pfam gathering cutoff for said protein domain families are stated in Table 17; wherein said plant cell nucleus is selected by screening a population of transgenic plants that have said recombinant DNA and express said protein for an enhanced trait as compared to control plants that do not have said recombinant DNA in their nuclei; and wherein said enhanced trait is selected from group of enhanced traits consisting of enhanced water use efficiency, enhanced cold tolerance, enhanced heat tolerance, enhanced resistance to salt exposure, enhanced shade tolerance, increased yield, enhanced nitrogen use efficiency, enhanced seed protein and enhanced seed oil.
 2. The plant cell nucleus of claim 1 wherein said protein has an amino acid sequence with at least 90% identity to a consensus amino acid sequence in the group of consensus amino acid sequences consisting of the consensus amino acid sequence constructed for SEQ ID NO: 198 and homologs thereof listed in Table 2 through the consensus amino acid sequence constructed for SEQ ID NO: 394 and homologs thereof listed in Table
 2. 3. The plant cell nucleus of claim 1 wherein said protein is selected from the group of proteins identified in Table
 1. 4. A plant cell nucleus with stably integrated, recombinant DNA to suppress the level of an endogenous protein having at least one domain of amino acids in a sequence that exceeds the Pfam gathering cutoff for amino acid sequence alignment with a protein domain family identified by Pfam name in the group of Pfam names consisting of SNF5, LMBR1, TFIIS_M, TFIIS_C, and Glyco_transf_(—)8, wherein the Pfam gathering cutoff for said protein domain families is stated in Table 17 wherein said plant cell nucleus is selected by screening a population of transgenic plants with said recombinant DNA and have the level of said endogenous protein suppressed for an enhanced trait as compared to control plants that do not have said recombinant DNA; and wherein said enhanced trait is selected from the group of enhanced traits consisting of enhanced water use efficiency, enhanced cold tolerance, enhanced heat tolerance, enhanced resistance to salt exposure, enhanced shade tolerance, increased yield, enhanced nitrogen use efficiency, enhanced seed protein and enhanced seed oil.
 5. The plant cell nucleus of claim 1 further comprising DNA expressing a protein that provides tolerance from exposure to an herbicide applied at levels that are lethal to a wild type of said plant cell.
 6. The plant cell nucleus of claim 5 wherein the agent of said herbicide is a glyphosate, dicamba, or glufosinate compound.
 7. A transgenic plant cell or plant comprising a plurality of plant cells with a plant cell nucleus of claim
 1. 8. The transgenic plant cell or plant of claim 7 which is homozygous for said recombinant DNA.
 9. A transgenic seed comprising a plurality of plant cells with a plant cell nucleus of claim
 1. 10. The transgenic seed of claim 9 from a corn, soybean, cotton, canola, alfalfa, wheat or rice plant.
 11. The transgenic corn seed of claim 10 wherein said seed can produce corn plants that are resistant to disease from the Mal de Rio Cuarto virus or the Puccina sorghi fungus or both.
 12. A transgenic pollen grain comprising a haploid derivative of a plant cell nucleus of claim
 1. 13. A method for manufacturing non-natural, transgenic seed that can be used to produce a crop of transgenic plants with an enhanced trait resulting from expression of stably-integrated recombinant DNA in a nucleus of claim 1, wherein said method for manufacturing said transgenic seed comprising: (a) screening a population of plants for said enhanced trait and said recombinant DNA, wherein individual plants in said population can exhibit said trait at a level less than, essentially the same as or greater than the level that said trait is exhibited in control plants which do not express the recombinant DNA, (b) selecting from said population one or more plants that exhibit said trait at a level greater than the level that said trait is exhibited in control plants, (c) verifying that said recombinant DNA is stably integrated in said selected plants, (d) analyzing tissue of said selected plant to determine the production or suppression of a protein having the function of a protein encoded by nucleotides having a sequence selected from the group consisting of one of SEQ ID NO:198-394; and (e) collecting seed from said selected plant.
 14. The method of claim 13 wherein plants in said population further comprise DNA expressing a protein that provides tolerance to exposure to an herbicide applied at levels that are lethal to wild type plant cells, and wherein said selecting is effected by treating said population with said herbicide.
 15. The method of claim 14 wherein said herbicide comprises a glyphosate, dicamba, or glufosinate compound.
 16. The method of claim 15 wherein said selecting is effected by identifying plants with said enhanced trait.
 17. The method of claim 16 wherein said seed is corn, soybean, cotton, alfalfa, wheat or rice seed.
 18. A method of producing hybrid corn seed comprising: (a) acquiring hybrid corn seed from a herbicide tolerant corn plant which also has stably-integrated, recombinant DNA in a nucleus of claim 1; (b) producing corn plants from said hybrid corn seed, wherein a fraction of the plants produced from said hybrid corn seed is homozygous for said recombinant DNA, a fraction of the plants produced from said hybrid corn seed is hemizygous for said recombinant DNA, and a fraction of the plants produced from said hybrid corn seed has none of said recombinant DNA; (c) selecting corn plants which are homozygous and hemizygous for said recombinant DNA by treating with an herbicide; (d) collecting seed from herbicide-treated-surviving corn plants and planting said seed to produce further progeny corn plants; (e) repeating steps (c) and (d) at least once to produce an inbred corn line; (f) crossing said inbred corn line with a second corn line to produce hybrid seed. 